Helical cytokine zalpha33

ABSTRACT

Novel cytokine polypeptides, materials and methods for making them, and method of use are disclosed. The polypeptides comprise at least nine contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4, and may be prepared as polypeptide fusions comprise heterologous sequences, such as affinity tags. The polypeptides and polynucleotides encoding them may be used within a variety of therapeutic, diagnostic, and research applications.

BACKGROUND OF THE INVENTION

[0001] Cytokines are polypeptide hormones that are produced by a celland affect the growth or metabolism of that cell or another cell. Inmulticellular animals, cytokines control cell growth, migration,differentiation, and maturation. Cytokines play a role in both normaldevelopment and pathogenesis, including the development of solid tumors.

[0002] Cytokines are physicochemically diverse, ranging in size from 5kDa (TGF-α) to 140 kDa (Mullerian-inhibiting substance). They includesingle polypeptide chains, as well as disulfide-linked homodimers andheterodimers.

[0003] Cytokines influence cellular events by binding to cell-surfacereceptors. Binding initiates a chain of signalling events within thecell, which ultimately results in phenotypic changes such as celldivision, protease production, cell migration, expression of cellsurface proteins, and production of additional growth factors.

[0004] Cell differentiation and maturation are also under control ofcytokines. For example, the hematopoietic factors erythropoietin,thrombopoietin, and G-CSF stimulate the production of erythrocytes,platelets, and neutrophils, respectively, from precursor cells in thebone marrow. Development of mature cells from pluripotent progenitorsmay require the presence of a plurality of factors.

[0005] The role of cytokines in controlling cellular processes makesthem likely candidates and targets for therapeutic intervention; indeed,a number of cytokines have been approved for clinical use.Interferon-alpha (IFN-α), for example, is used in the treatment of hairycell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, condylomataacuminata, chronic hepatitis C, and chronic hepatitis B (Aggarwal andPuri, “Common and Uncommon Features of Cytokines and Cytokine Receptors:An Overview”, in Aggarwal and Puri, eds., Human Cytokines: Their Role inDisease and Therapy, Blackwell Science, Cambridge, Mass., 1995, 3-24).Platelet-derived growth factor (PDGF) has been approved in the UnitedStates and other countries for the treatment of dermal ulcers indiabetic patients. The hematopoietic cytokine erythropoietin has beendeveloped for the treatment of anemias (e.g., EP 613,683). G-CSF,GM-CSF, IFN-β, IFN-γ, and IL-2 have also been approved for use in humans(Aggarwal and Puri, ibid.). Experimental evidence supports additionaltherapeutic uses of cytokines and their inhibitors. Inhibition of PDGFreceptor activity has been shown to reduce intimal hyperplasia ininjured baboon arteries (Giese et al., Restenosis Summit VIII, PosterSession #23, 1996; U.S. Pat. No. 5,620,687). Vascular endothelial growthfactors (VEGFs) have been shown to promote the growth of blood vesselsin ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and havebeen proposed for use as wound-healing agents, for treatment ofperiodontal disease, for promoting endothelialization in vascular graftsurgery, and for promoting collateral circulation following myocardialinfarction (WIPO Publication No. WO 95/24473; U.S. Pat. No. 5,219,739).A soluble VEGF receptor (soluble fit-1) has been found to block bindingof VEGF to cell-surface receptors and to inhibit the growth of vasculartissue in vitro (Biotechnology News 16(17):5-6, 1996). Experimentalevidence suggests that inhibition of angiogenesis may be used to blocktumor development (Biotechnology News, Nov. 13, 1997) and thatangiogenesis is an early indicator of cervical cancer (Br. J. Cancer76:1410-1415, 1997). More recently, thrombopoietin has been shown tostimulate the production of platelets in vivo (Kaushansky et al., Nature369:568-571, 1994) and has been the subject of several clinical trials(reviewed by von dem Borne et al., Bailliere 's Clin. Haematol.11:427-445, 1998).

[0006] In view of the proven clinical utility of cytokines, there is aneed in the art for additional such molecules for use as boththerapeutic agents and research tools and reagents. Cytokines are usedin the laboratory to study developmental processes, and in laboratoryand industry settings as components of cell culture media.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to provide novelpolypeptides, polynucleotides encoding them, and methods of making them.

[0008] It is another object of the invention to provide compositions andmethods for modulating the proliferation, differentiation, migration,and metabolism of responsive cell types and for regulating tissuedevelopment.

[0009] Within one aspect of the invention there is provided an isolatedpolypeptide comprising at least nine contiguous amino acid residues ofSEQ ID NO:2 or SEQ ID NO:4. Within one embodiment, the polypeptide hasfrom 15 to 1500 amino acid residues. Within another embodiment, thepolypeptide comprises at least nine contiguous amino acid residues ofSEQ ID NO:2 or SEQ ID NO:4 operably linked via a peptide bond orpolypeptide linker to a second polypeptide selected from the groupconsisting of maltose binding protein, an immunoglobulin constantregion, a polyhistidine tag, and a peptide as shown in SEQ ID NO:7.Within further embodiments, the polypeptide comprises at least 30contiguous residues of SEQ ID NO:2 or SEQ ID NO:4. Within otherembodiments, the polypeptide comprises residues 41-55 of SEQ ID NO:2,residues 56-77 of SEQ ID NO:2, residues 78-92 of SEQ ID NO:2, residues78-92 of SEQ ID NO:4, residues 93-110 of SEQ ID NO:2, residues 111-125of SEQ ID NO:2, residues 111-125 of SEQ ID NO:4, residues 126-148 of SEQID NO:2, residues 126-148 of SEQ ID NO:4, or residues 149-163 of SEQ IDNO:2. Within additional embodiments, the polypeptide comprises residues41-163 of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5; residues 34-163 ofSEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5; residues 34-178 of SEQ IDNO:2, SEQ ID NO:4, or SEQ ID NO:5; or residues 18-178 of SEQ ID NO: 2,SEQ ID NO:4, or SEQ ID NO:5.

[0010] Within a second aspect of the invention there is provided anexpression vector comprising the following operably linked elements: atranscription promoter, a DNA segment encoding a polypeptide asdisclosed above, and a transcription terminator. Within one embodiment,the DNA segment comprises nucleotides 52 to 534 of SEQ ID NO:6. Withinanother embodiment, the expression vector further comprises a secretorysignal sequence operably linked to the DNA segment.

[0011] Within a third aspect, the invention provides a cultured cellinto which has been introduced an expression vector as disclosed above,wherein the cell expresses the DNA segment. The cell can be used withina method of making a polypeptide, the method comprising culturing thecell under conditions whereby the DNA segment is expressed and thepolypeptide is produced, and recovering the polypeptide. Within oneembodiment, the expression vector further comprises a secretory signalsequence operably linked to the DNA segment, and the polypeptide issecreted by the cell and recovered from a medium in which the cell iscultured.

[0012] Within a further aspect of the invention there is provided apolypeptide produced by the method disclosed above.

[0013] Within another aspect, the invention provides an antibody thatspecifically binds to the polypeptide disclosed above.

[0014] Within an additional aspect, the invention provides a method ofdetecting, in a test sample, the presence of an antagonist of zalpha33activity. The method comprises the steps of (a) culturing a cell that isresponsive to zalpha33; (b) exposing the cell to a zalpha33 polypeptidein the presence and absence of a test sample; (c) comparing levels ofresponse to the zalpha33 polypeptide, in the presence and absence of thetest sample, by a biological or biochemical assay; and (d) determiningfrom the comparison the presence of an antagonist of zalpha33 activityin the test sample.

[0015] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a Hopp/Woods hydrophilicity profile of the amino acidsequence shown in SEQ ID NO:2. The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. These residues are indicated in the figure bylower case letters.

[0017]FIG. 2 shows an alignment of representative human (SEQ ID NO:2)and mouse (SEQ ID NO:4) zalpha33 polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0019] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985) (SEQID NO:7), substance P, Flag™ peptide (Hopp et al., Biotechnology6:1204-1210, 1988), streptavidin binding peptide, maltose bindingprotein (Guan et al., Gene 67:21-30, 1987), cellulose binding protein,thioredoxin, ubiquitin, T7 polymerase, or other antigenic epitope orbinding domain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags and otherreagents are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, N.J.; New England Biolabs, Beverly, Mass.; EastmanKodak, New Haven, Conn.).

[0020] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0021] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0022] A “complement” of a polynucleotide molecule is a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence. For example, the sequence 5′ ATGCACGGG3′ is complementary to 5′CCCGTGCAT 3′.

[0023] The term “corresponding to”, when applied to positions of aminoacid residues in sequences, means corresponding positions in a pluralityof sequences when the sequences are optimally aligned.

[0024] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0025] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0026] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural geneticmilieu-and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, but mayinclude naturally occurring 5′ and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art (see for example, Dynanand Tijan, Nature 316:774-78, 1985).

[0027] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. The isolated polypeptide or proteinmay be substantially free of other polypeptides or proteins,particularly those of animal origin. An isolated polypeptide or proteinmay be provided in a highly purified form, i.e.greater than 95% pure orgreater than 99% pure. When used in this context, the term “isolated”does not exclude the presence of the same polypeptide or protein inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0028] “Operably linked” means that two or more entities are joinedtogether such that they function in concert for their intended purposes.When referring to DNA segments, the phrase indicates, for example, thatcoding sequences are joined in the correct reading frame, andtranscription initiates in the promoter and proceeds through the codingsegment(s) to the terminator. When referring to polypeptides, “operablylinked” includes both covalently (e.g., by disulfide bonding) andnon-covalently (e.g., by hydrogen bonding, hydrophobic interactions, orsalt-bridge interactions) linked sequences, wherein the desiredfunction(s) of the sequences are retained.

[0029] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0030] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be. recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

[0031] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0032] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0033] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0034] A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

[0035] A “segment” is a portion of a larger molecule (e.g.,polynucleotide or polypeptide) having specified attributes. For example,a DNA segment encoding a specified polypeptide is a portion of a longerDNA molecule, such as a plasmid or plasmid fragment, that, when readfrom the 5′ to the 3′ direction, encodes the sequence of amino acids ofthe specified polypeptide.

[0036] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0037] All references cited herein are incorporated by reference intheir entirety.

[0038] The present invention provides novel cytokine polypeptides andproteins. This novel cytokine, termed “zalpha33”, was identified by thepresence of polypeptide and polynucleotide features characteristic offour-helix-bundle cytokines (e.g., erythropoietin, thrombopoietin,G-CSF, IL-2, IL4, leptin, and growth hormone). Analysis of the aminoacid sequence shown in SEQ ID NO:2 indicates the presence of fouramphipathic, alpha-helical regions. These regions include at least aminoacid residues 41 through 55 (helix A), 78 through 92 (helix B), 111through 125 (helix C), and 149 through 163 (helix D). Within thesehelical regions, residues that are expected to lie within the core ofthe four-helix bundle occur at positions 41, 44, 45, 48, 51, 52, 55, 78,81, 82, 85, 88, 89, 92, 111, 114, 115, 118, 121, 122, 125, 149, 152,153, 156, 159, 160, and 163 of SEQ ID NO:2. Residues 42, 43, 46, 47, 49,50, 53, 54, 79, 80, 83, 84, 86, 87, 90, 91, 112, 113, 116, 117, 119,120, 123, 124, 150, 151, 154, 155, 157, 158, 161, and 162 are expectedto lie on the exposed surface of the bundle. Inter-helix loops compriseapproximately residues 56 through 77 (loop A-B), 93 through 110 (loopB-C), and 126 through 148 (loop C-D). The human zalpha33 cDNA (SEQ IDNO: 1) encodes a polypeptide of 178 amino acid residues. While notwishing to be bound by theory, this sequence is predicted to include asecretory peptide of 17 residues. Cleavage after residue 17 will resultin a mature polypeptide (residues 18-178 of SEQ ID NO:2) having acalculated molecular weight (exclusive of glycosylation) of 18,655 Da.Those skilled in the art will recognize, however, that some cytokines(e.g., endothelial cell growth factor, basic FGF, and IL-1β) do notcomprise conventional secretory peptides and are secreted by a mechanismthat is not understood. The cDNA also includes a clear polyadenylationsignal, as well as two message instability motifs (ATTTA) in the3′-untranslated region beginnning at nucleotides 679 and 753. Thesemessage instability motifs are characteristic of cytokine genes (see,e.g., Shaw and Kamen, Cell 46:659-667, 1986).

[0039] Those skilled in the art will recognize that predicted domainboundaries are somewhat imprecise and may vary by up to ±5 amino acidresidues.

[0040] Polypeptides of the present invention comprise at least 6, atleast 9, or at least 15 contiguous amino acid residues of SEQ ID NO:2.Within certain embodiments of the invention, the polypeptides comprise20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO:2, up tothe entire predicted mature polypeptide (residues 18 to 178 of SEQ IDNO:2) or the primary translation product (residues 1 to 178 of SEQ IDNO:2). As disclosed in more detail below, these polypeptides can furthercomprise additional, non-zalpha33, polypeptide sequence(s).Corresponding mouse zalpha33 polypeptides (see SEQ ID NO:4) are alsoprovided by the invention.

[0041] Within the polypeptides of the present invention are polypeptidesthat comprise an epitope-bearing portion of a protein as shown in SEQ IDNO:2 or SEQ ID NO:4. An “epitope” is a region of a protein to which anantibody can bind. See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002, 1984. Epitopes can be linear or conformational,the latter being composed of discontinuous regions of the protein thatform an epitope upon folding of the protein. Linear epitopes aregenerally at least 6 amino acid residues in length. Relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, Sutcliffe et al., Science 219:660-666, 1983.Antibodies that recognize short, linear epitopes are particularly usefulin analytic and diagnostic applications that employ denatured protein,such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA76:4350-4356, 1979), or in the analysis of fixed cells or tissuesamples. Antibodies to linear epitopes are also useful for detectingfragments of zalpha33, such as might occur in body fluids or cellculture media.

[0042] Antigenic, epitope-bearing polypeptides of the present inventionare useful for raising antibodies, including monoclonal antibodies, thatspecifically bind to a zalpha33 protein. Antigenic, epitope-bearingpolypeptides contain a sequence of at least six, often at least nine,commonly from 15 to about 30 contiguous amino acid residues of azalpha33 protein (e.g., SEQ ID NO:2). Polypeptides comprising a largerportion of a zalpha33 protein, i.e. from 30 to 50 residues up to theentire sequence, are included. It is preferred that the amino acidsequence of the epitope-bearing polypeptide is selected to providesubstantial solubility in aqueous solvents, that is the sequenceincludes relatively hydrophilic residues, and hydrophobic residues aresubstantially avoided. Such regions include the interdomain loops ofzalpha33 and fragments thereof, particular loop C-D, which is markedlyhydrophilic (see FIG. 1). Exemplary polypeptides in this regard includethose comprising residues 136-141, 123-128, 119-124, 135-140, or 147-152of SEQ ID NO:2.

[0043] Of particular interest within the present invention arepolypeptides that comprise the entire four-helix bundle of a zalpha33polypeptide (e.g., residues 41-163 of SEQ ID NO:2). Such polypeptidesmay further comprise all or part of one or both of the native zalpha33amino-terminal (residues 18-40 of SEQ ID NO:2) and carboxyl-terminal(residues 164-178 of SEQ ID NO:2) regions, as well as non-zalpha33 aminoacid residues or polypeptide sequences as disclosed in more detailbelow.

[0044] Polypeptides of the present invention can be prepared with one ormore amino acid substitutions, deletions or additions as compared to SEQID NO:2. These changes will usually be of a minor nature, that isconservative amino acid substitutions and other changes that do notsignificantly affect the folding or activity of the protein orpolypeptide, and include amino- or carboxyl-terminal extensions, such asan amino-terminal methionine residue, an amino or carboxyl-terminalcysteine residue to facilitate subsequent linking to maleimide-activatedkeyhole limpet hemocyanin, a small linker peptide of up to about 20-25residues, or an extension that facilitates purification (an affinitytag) as disclosed above. Two or more affinity tags may be used incombination. Polypeptides comprising affinity tags can further comprisea polypeptide linker and/or a proteolytic cleavage site between thezalpha33 polypeptide and the affinity tag. Exemplary cleavage sitesinclude thrombin cleavage sites and factor Xa cleavage sites.

[0045] The present invention further provides a variety of otherpolypeptide fusions. For example, a zalpha33 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Suitable dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-zalpha33polypeptide fusions can be expressed in genetically engineered cells toproduce a variety of multimeric zalpha33 analogs. In addition, azalpha33 polypeptide can be joined to another bioactive molecule, suchas a cytokine, to provide a multi-functional molecule. One or morehelices of a zalpha33 polypeptide can be joined to another cytokine toenhance or otherwise modify its biological properties. Auxiliary domainscan be fused to zalpha33 polypeptides to target them to specific cells,tissues, or macromolecules (e.g., collagen). For example, a zalpha33polypeptide or protein can be targeted to a predetermined cell type byfusing a zalpha33 polypeptide to a ligand that specifically binds to areceptor on the surface of the target cell. In this way, polypeptidesand proteins can be targeted for therapeutic or diagnostic purposes. Azalpha33 polypeptide can be fused to two or more moieties, such as anaffinity tag for purification and a targeting domain. Polypeptidefusions can also comprise one or more cleavage sites, particularlybetween domains. See, Tuan et al., Connective Tissue Research 34:1-9,1996.

[0046] Polypeptide fusions of the present invention will generallycontain not more than about 1,500 amino acid residues, often not morethan about 1,200 residues, -frequently not more than about 1,000residues, and will in many cases be considerably smaller. For example, azalpha33 polypeptide of 161 residues (residues 18-178 of SEQ ID NO:2)can be fused to E. coli β-galactosidase (1,021 residues; see Casadabanet al., J. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a4-residue factor Xa cleavage site to yield a polypeptide of 1,196residues. In a second example, residues 18-178 of SEQ ID NO:2 can befused to maltose binding protein (approximately 370 residues), a4-residue cleavage site, and a 6-residue polyhistidine tag.

[0047] As disclosed above, the polypeptides of the present inventioncomprise at least 6 contiguous residues of SEQ ID NO:2 or SEQ ID NO:4.These polypeptides may further comprise additional residues as shown inSEQ ID NO:2, a variant of SEQ ID NO:2, or another protein as disclosedherein. “Variants of SEQ ID NO:2” includes polypeptides that are atleast 90% or at least 95% identical to the corresponding region of SEQID NO:2. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616,1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919, 1992. Briefly, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 1, and the “BLOSUM62” scoring matrix of Henikoffand Henikoff (ibid.) as shown in Table 1 (amino acids are indicated bythe standard one-letter codes). The percent identity is then calculatedas:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}\quad {number}} \right. \\{{of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}\quad {sequence}\quad {in}} \\\left. {{order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {{sequences}.}} \right\rbrack\end{matrix}} \times 100$

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 1 1 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0048] The level of identity between amino acid sequences can bedetermined using the “FASTA” similarity search algorithm disclosed byPearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988) and byPearson (Meth. Enzymol. 183:63, 1990). Briefly, FASTA firstcharacterizes sequence similarity by identifying regions shared by thequery sequence (e.g., SEQ ID NO:2) and a test sequence that have eitherthe highest density of identities (if the ktup variable is 1) or pairsof identities (if ktup=2), without considering conservative amino acidsubstitutions, insertions, or deletions. The ten regions with thehighest density of identities are then rescored by comparing thesimilarity of all paired amino acids using an amino acid substitutionmatrix, and the ends of the regions are “trimmed” to include only thoseresidues that contribute to the highest score. If there are severalregions with scores greater than the “cutoff” value (calculated by apredetermined formula based upon the length of the sequence and the ktupvalue), then the trimmed initial regions are examined to determinewhether the regions can be joined to form an approximate alignment withgaps. Finally, the highest scoring regions of the two amino acidsequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup-1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, 1990 (ibid.).

[0049] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from three to six, most preferably three, with otherparameters set as default.

[0050] The present invention includes polypeptides having one or moreconservative amino acid changes as compared with the amino acid sequenceof SEQ ID NO:2. The BLOSUM62 matrix (Table 1) is an amino acidsubstitution matrix derived from about 2,000 local multiple alignmentsof protein sequence segments, representing highly conserved regions ofmore than 500 groups of related proteins (Henikoff and Henikoff, ibid.).Thus, the BLOSUM62 substitution frequencies can be used to defineconservative amino acid substitutions that may be introduced into theamino acid sequences of the present invention. As used herein, the term“conservative amino acid substitution” refers to a substitutionrepresented by a BLOSUM62 value of greater than −1. For example, anamino acid substitution is conservative if the substitution ischaracterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least one 1 (e.g., 1, 2 or 3), while more preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 2 (e.g., 2 or 3).

[0051] The proteins of the present invention can also comprisenon-naturally occuring amino acid residues. Non-naturally occuring aminoacids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occuring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating TRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-19998, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccuring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,-4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccuring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occuring amino acid residues can be converted to non-naturallyoccuring species by in vitro chemical modification. Chemicalmodification can be combined with site-directed mutagenesis to furtherexpand the range of substitutions (Wynn and Richards, Protein Sci.2:395-403, 1993).

[0052] Amino acid sequence changes are made in zalpha33 polypeptides soas to minimize disruption of higher order structure essential tobiological activity. For example, changes in amino acid residues will bemade so as not to disrupt the four-helix bundle characteristic of theprotein family. The effects of amino acid sequence changes can bepredicted by computer modeling using available software (e.g., theInsight II viewer and homology modeling tools; MSI, San Diego, Calif.)or determined by analysis of crystal structure (see, e.g., Lapthom etal., Nature 369:455-461, 1994 and Lapthorn et al., Nat. Struct. Biol.2:266-268, 1995). A hydrophilicity profile of SEQ ID NO:2 is shown inFIG. 1. Those skilled in the art will recognize that this hydrophilicitywill be taken into account when designing alterations in the amino acidsequence of a zalpha33 polypeptide, so as not to disrupt the overallprofile. Residues within the core of the four-helix bundle can bereplaced with a hydrophobic residue selected from the group consistingof Leu, Ile, Val, Met, Phe, Trp, Gly, and Ala as shown in SEQ ID NO:5.Cysteine residues at positions 99 and 103 of SEQ ID NO:2 and SEQ ID NO:4and the residues predicted to be on the exposed surface of thefour-helix bundle will be relatively intolerant of substitution. Othercandidate amino acid substitutions within human zalpha33 are suggestedby alignment of the human (SEQ ID NO:2) and mouse (SEQ ID NO:4)sequences as shown in FIG. 2, which sequences are approximately 95%identical overall.

[0053] One skilled in the art may employ many well known techniques,independently or in combination, to analyze and compare the structuralfeatures that affect folding of a variant protein or polypeptide to astandard molecule to determine whether such modifications would besignificant. One well known and accepted method for measuring folding iscircular dichroism (CD). Measuring and comparing the CD spectragenerated by a modified molecule and standard molecule are routine inthe art (Johnson, Proteins 7:205-214, 1990). Crystallography is anotherwell known and accepted method for analyzing folding and structure.Nuclear magnetic resonance (NMR), digestive peptide mapping and epitopemapping are other known methods for analyzing folding and structurallysimilarities between proteins and polypeptides (Schaanan et al., Science257:961-964, 1992).

[0054] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-4502, 1991). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule.

[0055] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0056] Variants of the disclosed zalpha33 DNA and polypeptide sequencescan be generated through DNA shuffling as disclosed by Stemmer, Nature370:389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA91:10747-10751, 1994. Briefly, variant genes are generated by in vitrohomologous recombination by random fragmentation of a parent genefollowed by reassembly using PCR, resulting in randomly introduced pointmutations. This technique can be modified by using a family of parentgenes, such as allelic variants or genes from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0057] In many cases, the structure of the final polypeptide productwill result from processing of the nascent polypeptide chain by the hostcell, thus the final sequence of a zalpha33 polypeptide produced by ahost cell will not always correspond to the full sequence encoded by theexpressed polynucleotide. For example, expressing the complete zalpha33sequence in a cultured mammalian cell is expected to result in removalof at least the secretory peptide, while the same polypeptide producedin a prokaryotic host would not be expected to be cleaved. Differentialprocessing of individual chains may result in heterogeneity of expressedpolypeptides.

[0058] The human and mouse zalpha33 polypeptide sequences each contain 3cysteine residues, at positions 34, 99, and 103. Structural predictionsindicate that Cys99 and Cys103 may form an intrachain disulfide bond,and that Cys34 may be free to form an interchain disulfide bond,resulting in dimerization. Actual conformation will depend in part uponthe cell in which in the polypeptide is expressed. The polypeptides ofthe present invention thus include those comprising these cysteineresidues, such as polypeptides comprising residues 34-163 or 34-178 ofSEQ ID NO:2 or SEQ ID NO:4.

[0059] Zalpha33 proteins of the present invention are characterized bytheir activity, that is, modulation of the proliferation,differentiation, migration, adhesion, or metabolism of responsive celltypes. Responsive cell types include epithelial cells and the cell linesU-373 MG (human brain glioblastoma) and 3A-Sub E (SV40-transformed humanplacenta). Biological activity of zalpha33 proteins is assayed using invitro or in vivo assays designed to detect cell proliferation,differentiation, migration or adhesion; or changes in cellularmetabolism (e.g., production of other growth factors or othermacromolecules). Many suitable assays are known in the art, andrepresentative assays are disclosed herein. Assays using cultured cellsare most convenient for screening, such as for determining the effectsof amino acid substitutions, deletions, or insertions. However, in viewof the complexity of developmental processes (e.g., angiogenesis, woundhealing), in vivo assays will generally be employed to confirm andfurther characterize biological activity. Certain in vitro models, suchas the three-dimensional collagen gel matrix model of Pepper et al.(Biochem. Biophys. Res. Comm. 189:824-831, 1992), are sufficientlycomplex to assay histological effects. Assays can be performed usingexogenously produced proteins, or may be carried out in vivo or in vitrousing cells expressing the polypeptide(s) of interest. Assays can beconducted using zalpha33 proteins alone or in combination with othergrowth factors, such as members of the VEGF family or hematopoieticcytokines (e.g., EPO, TPO, G-CSF, stem cell factor). Representativeassays are disclosed below.

[0060] Mutagenesis methods as disclosed above can be combined with highvolume or high-throughput screening methods to detect biologicalactivity of zalpha33 variant polypeptides. Assays that can be scaled upfor high throughput include mitogenesis assays, which can be run in a96-well format. Mutagenized DNA molecules that encode active zalpha33polypeptides can be recovered from the host cells and rapidly sequencedusing modern equipment. These methods allow the rapid determination ofthe importance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

[0061] Using the methods discussed above, one of ordinary skill in theart can prepare a variety of polypeptide fragments or variants of SEQ IDNO:2 or SEQ ID NO:4 that retain the activity of wild-type zalpha33.

[0062] The present invention also provides polynucleotide molecules,including DNA and RNA molecules, that encode the zalpha33 polypeptidesdisclosed above. A representative DNA sequence encoding the amino acidsequence of SEQ ID NO:2 is shown in SEQ ID NO: 1, and a representativeDNA sequence encoding the amino acid sequence of SEQ ID NO:4 is shown inSEQ ID NO:3. Those skilled in the art will readily recognize that, inview of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:6is a degenerate DNA sequence that encompasses all DNAs that encode thezalpha33 polypeptide of SEQ ID NO: 2. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:6 also provides allRNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus,zalpha33 polypeptide-encoding polynucleotides comprising nucleotides1-534 or nucleotides 52-534 of SEQ ID NO:6, and their RNA equivalentsare contemplated by the present invention, as are segments of SEQ IDNO:6 encoding other zalpha33 polypeptides disclosed herein. Table 2 setsforth the one-letter codes used within SEQ ID NO:6 to denote degeneratenucleotide positions. “Resolutions” are the nucleotides denoted by acode letter. “Complement” indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C or T, and itscomplement R denotes A or G. A being complementary to T, and G beingcomplementary to C. TABLE 2 Nucleotide Resolutions ComplementResolutions A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G MA|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T VA|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T

[0063] The degenerate codons used in SEQ ID NO:6, encompassing allpossible codons for a given amino acid, are set forth in Table 3, below.TABLE 3 One- Amino Letter Degenerate Acid Code Codons Codon Cys C TGCTGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT CAN ProP CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGTGGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAGCAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAGAAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTGYTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp WTGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN Gap— —

[0064] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NO: 2. Variant sequences can bereadily tested for functionality as described herein.

[0065] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit preferential codon usage. See, in general,Grantham et al., Nuc. Acids Res. 8:1893-912, 1980; Haas et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson et al., Gene 13:355-64, 1981; Grosjeanand Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87,1986; and Ikemura, J. Mol. Biol. 158:573-97, 1982. Introduction ofpreferred codon sequences into recombinant DNA can, for example, enhanceproduction of the protein by making protein translation more efficientwithin a particular cell type or species. Therefore, the degeneratecodon sequence disclosed in SEQ ID NO:6 serves as a template foroptimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein.

[0066] Within certain embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1or SEQ ID NO:3, or a sequence complementary thereto, under stringentconditions. In general, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typicalstringent conditions are those in which the salt concentration is up toabout 0.03 M at pH 7 and the temperature is at least about 60° C.

[0067] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zalpha33 RNA. Suitable tissues includeheart, liver, pancreas, testis, ovary, and thyroid. Zalpha33 transcriptshave also been detected in B-cells and in many tumor cells. Total RNAcan be prepared using guanidine HCl extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412,1972). Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA usingknown methods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding zalpha33 polypeptides are then identified andisolated by, for example, hybridization or PCR.

[0068] Full-length clones encoding zalpha33 can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones areoften preferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to zalpha33, receptor fragments,or other specific binding partners.

[0069] Zalpha33 polynucleotide sequences disclosed herein can also beused as probes or primers to clone 5′ non-coding regions of a zalpha33gene. Promoter elements from a zalpha33 gene could thus be used todirect the expression of heterologous genes in, for example, transgenicanimals or patients treated with gene therapy. Cloning of 5′ flankingsequences also facilitates production of zalpha33 proteins by “geneactivation” as disclosed in U.S. Pat. No. 5,641,670. Briefly, expressionof an endogenous zalpha33 gene in a cell is altered by introducing intothe zalpha33 locus a DNA construct comprising at least a targetingsequence, a regulatory sequence, an exon, and an unpaired splice donorsite. The targeting sequence is a zalpha33 5′ non-coding sequence thatpermits homologous recombination of the construct with the endogenouszalpha33 locus, whereby the sequences within the construct becomeoperably linked with the endogenous zalpha33 coding sequence. In thisway, an endogenous zalpha33 promoter can be replaced or supplementedwith other regulatory sequences to provide enhanced, tissue-specific, orotherwise regulated expression.

[0070] A human zalpha33 gene sequence is shown in SEQ ID NO: 10. WithinSEQ ID NO:10, exons are at nucleotides 1001-1319, 8914-9072,10,986-11,090, and 20,144-20,597. Nucleotides 1-1000 of SEQ ID NO:2 arebelieved to contain promoter and regulatory elements.

[0071] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NOS: 1 and 2 represent a single allele of humanzalpha33, and the sequences disclosed in SEQ ID NOS:3 and 4 represent asingle allele of mouse zalpha33. Allelic variants of these sequences canbe cloned by probing cDNA or genomic libraries from differentindividuals according to standard procedures.

[0072] The present invention further provides counterpart polypeptidesand polynucleotides from other species (“orthologs”). Of particularinterest are zalpha33 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human zalpha33 can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using MRNA obtained from a tissue or cell type thatexpresses zalpha33 as disclosed above. A library is then prepared frommRNA of a positive tissue or cell line. A zalpha33-encoding cDNA canthen be isolated by a variety of methods, such as by probing with acomplete or partial human cDNA or with one or more sets of degenerateprobes based on the disclosed sequence. A cDNA can also be cloned usingthe polymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the representative human zalpha33 sequencedisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to zalpha33 polypeptide.Similar techniques can also be applied to the isolation of genomicclones.

[0073] For any zalpha33 polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 3 and 4, above. Moreover, those of skillin the art can use standard software to devise zalpha33 variants basedupon the nucleotide and amino acid sequences described herein. Thepresent invention thus provides a computer-readable medium encoded witha data structure that provides at least one of the following sequences:SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, and portions thereof. Suitable forms of computer-readable mediainclude magnetic media and optically-readable media. Examples ofmagnetic media include a hard or fixed drive, a random access memory(RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, anda ZIP™ disk. Optically readable media are exemplified by compact discs(e.g., CD-read only memory (ROM), CD-rewritable (RW), andCD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM,DVD-RAM, and DVD+RW).

[0074] The zalpha33 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides can be produced according to conventional techniques usingcells into which have been introduced an expression vector encoding thepolypeptide. As used herein, “cells into which have been introduced anexpression vector” include both cells that have been directlymanipulated by the introduction of exogenous DNA molecules and progenythereof that contain the introduced DNA. Suitable host cells are thosecell types that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al., eds., Current Protocols in Molecular Biology,John Wiley and Sons, Inc., NY, 1987.

[0075] In general, a DNA sequence encoding a zalpha33 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0076] To direct a zalpha33 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zalpha33, or may be derivedfrom another secreted protein (e.g., t-PA; see, U.S. Pat. No. 5,641,655)or synthesized de novo. The secretory signal sequence is operably linkedto the zalpha33 DNA sequence, i.e., the two sequences are joined in thecorrect reading frame and positioned to direct the newly sythesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

[0077] Cultured mammalian cells can be used as hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed, for example, by Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61; or CHODG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. In general, strong transcription promoters are will beused, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S.Pat. No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and theadenovirus major late promoter. Expression vectors for use in mammaliancells include pZP-1 and pZP-9, which have been deposited with theAmerican Type Culture Collection, Manassas, Va. USA under accessionnumbers 98669 and 98668, respectively, and derivatives thereof.

[0078] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Anexemplary selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.An exemplary amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

[0079] The adenovirus system (disclosed in more detail below) can alsobe used for protein production in vitro. By culturingadenovirus-infected non-293 cells under conditions where the cells arenot rapidly dividing, the cells can produce proteins for extendedperiods of time. For instance, BHK cells are grown to confluence in cellfactories, then exposed to the adenoviral vector encoding the secretedprotein of interest. The cells are then grown under serum-freeconditions, which allows infected cells to survive for several weekswithout significant cell division. In an alternative method, adenovirusvector-infected 293 cells can be grown as adherent cells or insuspension culture at relatively high cell density to producesignificant amounts of protein (See Gamier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins can also be effectivelyobtained.

[0080] Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV) according to methods known in the art. Within one method,recombinant baculovirus is produced through the use of atransposon-based system described by Luckow et al. (J. Virol.67:4566-4579, 1993). This system, which utilizes transfer vectors, iscommercially available in kit form (Bac-to-Bac™ kit; Life Technologies,Rockville, Md.). The transfer vector (e.g., pFastBac1™; LifeTechnologies) contains a Tn7 transposon to move the DNA encoding theprotein of interest into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen.Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556,1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.In addition, transfer vectors can include an in-frame fusion with DNAencoding a polypeptide extension or affinity tag as disclosed above.Using techniques known in the art, a transfer vector containing azalpha33-encoding sequence is transformed into E. coli host cells, andthe cells are screened for bacmids which contain an interrupted lacZgene indicative of recombinant baculovirus. The bacmid DNA containingthe recombinant baculovirus genome is isolated, using common techniques,and used to transfect Spodoptera frugiperda cells, such as Sf9 cells.Recombinant virus that expresses zalpha33 protein is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

[0081] For protein production, the recombinant virus is used to infecthost cells, typically a cell line derived from the fall armyworm,Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni(e.g., High Five™ cells; Invitrogen, Carlsbad, Calif.). See, forexample, U.S. Pat. No. 5,300,435. Serum-free media are used to grow andmaintain the cells. Suitable media formulations are known in the art andcan be obtained from commercial suppliers. The cells are grown up froman inoculation density of approximately 2-5×10⁵ cells to a density of1-2×10⁶ cells, at which time a recombinant viral stock is added at amultiplicity of infection (MOI) of 0.1 to 10, more typically near 3.Procedures used are generally known in the art.

[0082] Other higher eukaryotic cells can also be used as hosts,including plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.

[0083] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). An exemplary vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; andRaymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production ofrecombinant proteins in Pichia methanolica is disclosed in U.S. Pat.Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

[0084] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a zalpha33polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0085] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. Liquid culturesare provided with sufficient aeration by conventional means, such asshaking of small flasks or sparging of fermentors.

[0086] The polypeptides and proteins of the present invention can bepurified to ≧80% purity, to ≧90% purity, to ≧95% purity, or to apharmaceutically pure state, that is greater than 99.9% pure withrespect to contaminating macromolecules, particularly other proteins andnucleic acids, and free of infectious and pyrogenic agents. The desireddegree of purification will depend upon the intended use of thepolypeptide or protein.

[0087] Expressed recombinant zalpha33 proteins (including chimericpolypeptides and multimeric proteins) are purified by conventionalprotein purification methods, typically by a combination ofchromatographic techniques. See, in general, Affinity Chromatography:Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden,1988; and Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York, 1994. Proteins comprising a polyhistidineaffinity tag (typically about 6 histidine residues) are purified byaffinity chromatography on a nickel chelate resin. See, for example,Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising aglu-glu tag can be purified by immunoaffinity chromatography accordingto conventional procedures. See, for example, Grussenmeyer et al., ibid.Maltose binding protein fusions are purified on an amylose columnaccording to methods known in the art.

[0088] Zalpha33 polypeptides can also be prepared through chemicalsynthesis according to methods known in the art, including exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989. In vitro synthesis is particularly advantageous for thepreparation of smaller polypeptides.

[0089] PEGylation is one method commonly used that has been demonstratedto increase plasma half-life, increase solubility, and decreaseantigenicity and immunogenicity of proteins (Nucci et al., Advanced DrugDelivery Reviews 6:133-155, 1991 and Lu et al., Int. J. Peptide ProteinRes. 43:127-138, 1994). Several procedures for creating and purifyingpegylated proteins are known in the art. See, for example, Abuchowski etal., J. Biol. Chem. 252:3582-3586, 1977 and Becauchamp et al., Anal.Biochem. 131:25-33, 1983. Particular acid amino.acid residues (e.g.glutamic and aspartic acids) and amino acids at the carboxyl-termiinusof a protein are amenable to PEGylation (Zalipsky, Bioconjugate Chem.6:150-165, 1995).

[0090] Using methods known in the art, zalpha33 proteins can be preparedas monomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

[0091] Target cells for use in zalpha33 activity assays include, withoutlimitation, vascular cells (especially endothelial cells and smoothmuscle cells), hematopoietic (myeloid and lymphoid) cells, liver cells(including hepatocytes, fenestrated endothelial cells, Kupffer cells,and Ito cells), fibroblasts (including human dermal fibroblasts and lungfibroblasts), fetal lung cells, articular synoviocytes, pericytes,chondrocytes, osteoblasts, and prostate epithelial cells. Endothelialcells and hematopoietic cells are derived from a common ancestral cell,the hemangioblast (Choi et al., Development 125:725-732, 1998).

[0092] Activity of zalpha33 proteins can be measured in vitro usingcultured cells or in vivo by administering molecules of the claimedinvention to an appropriate animal model. Assays measuring cellproliferation or differentiation are well known in the art. For example,assays measuring proliferation include such assays as chemosensitivityto neutral red dye (Cavanaugh et al., Investigational New Drugs8:347-354, 1990), incorporation of radiolabelled nucleotides (asdisclosed by, e.g., Raines and Ross, Methods Enzymol. 109:749-773, 1985;Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988; and Cook et al.,Analytical Biochem. 179:1-7, 1989), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use oftetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley etal., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84,1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988).Differentiation can. be assayed using suitable precursor cells that canbe induced to differentiate into a more mature phenotype. Assaysmeasuring differentiation include, for example, measuring cell-surfacemarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference).

[0093] Zalpha33 activity may also be detected using assays designed tomeasure zalpha33-induced production of one or more additional growthfactors or other macromolecules. Such assays include those fordetermining the presence of hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), transforming growth factor alpha (TGFα),interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF),angiogenin, and other macromolecules produced by the liver. Suitableassays include mitogenesis assays using target cells responsive to themacromolecule of interest, receptor-binding assays, competition bindingassays, immunological assays (e.g., ELISA), and other formats known inthe art. Metalloprotease secretion is measured from treated primaryhuman dermal fibroblasts, synoviocytes and chondrocytes. The relativelevels of collagenase, gelatinase and stromalysin produced in responseto culturing in the presence of a zalpha33 protein is measured usingzymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106,1990). Procollagen/collagen synthesis by dermal fibroblasts andchondrocytes in response to a test protein is measured using ³H-prolineincorporation into nascent secreted collagen. ³H-labeled collagen isvisualized by SDS-PAGE followed by autoradiography (Unemori and Amento,J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG)secretion from dermal fibroblasts and chondrocytes is measured using a1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim.Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are alsocarried out in the presence of IL-1β or TGF-β to examine the ability ofzalpha33 protein to modify the established responses to these cytokines.

[0094] Monocyte activation assays are carried out (1) to look for theability of zalpha33 proteins to further stimulate monocyte activation,and (2) to examine the ability of zalpha33 proteins to modulateattachment-induced or endotoxin-induced monocyte activation (Fuhlbriggeet al., J. Immunol. 138: 3799-3802, 1987). IL-1β and TNFα levelsproduced in response to activation are measured by ELISA (Biosource,Inc. Camarillo, Calif.). Monocyte/macrophage cells, by virtue of CD14(LPS receptor), are exquisitely sensitive to endotoxin, and proteinswith moderate levels of endotoxin-like activity will activate thesecells.

[0095] Hematopoietic activity of zalpha33 proteins can be assayed onvarious hematopoietic cells in culture. Suitable assays include primarybone marrow colony assays and later stage lineage-restricted colonyassays, which are known in the art (e.g., Holly et al., WIPO PublicationWO 95/21920). Marrow cells plated on a suitable semi-solid medium (e.g.,50% methylcellulose containing 15% fetal bovine serum, 10% bovine serumalbumin, and 0.6% PSN antibiotic mix) are incubated in the presence oftest polypeptide, then examined microscopically for colony formation.Known hematopoietic factors are used as controls. Mitogenic activity ofzalpha33 polypeptides on hematopoietic cell lines can be measured asdisclosed above.

[0096] Cell migration is assayed essentially as disclosed by Kahler etal. (Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939,1997). A protein is considered to be chemotactic if it induces migrationof cells from an area of low protein concentration to an area of highprotein concentration. A typical assay is performed using modifiedBoyden chambers with a polystryrene membrane separating the two chambers(e.g., Transwell®; Corning Costar Corp.). The test sample, diluted inmedium containing 1% BSA, is added to the lower chamber of a 24-wellplate containing Transwells. Cells are then placed on the Transwellinsert that has been pretreated with 0.2% gelatin. Cell migration ismeasured after 4 hours of incubation at 37° C. Non-migrating cells arewiped off the top of the Transwell membrane, and cells attached to thelower face of the membrane are fixed and stained with 0.1% crystalviolet. Stained cells are then extracted with 10% acetic acid andabsorbance is measured at 600 nm. Migration is then calculated from astandard calibration curve. Cell migration can also be measured usingthe matrigel method of Grant et al. (“Angiogenesis as a component ofepithelial-mesenchymal interactions” in Goldberg and Rosen,Epithelial-Mesenchymal Interaction in Cancer, Birkhäuser Verlag, 1995,235-248; Baatout, Anticancer Research 17:451-456, 1997).

[0097] Growth factor effects of zalpha33 proteins can also be assayed inan aortic ring outgrowth assay (Nicosia and Ottinetti, LaboratoryInvestigation 63:115, 1990; Villaschi and Nicosia, Am. J. Pathology143:181-190, 1993).

[0098] Cell adhesion activity is assayed essentially as disclosed byLaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly,microtiter plates are coated with the test protein, non-specific sitesare blocked with BSA, and cells (such as smooth muscle cells,leukocytes, or endothelial cells) are plated at a density ofapproximately 10⁴-10⁵ cells/well. The wells are incubated at 37° C.(typically for about 60 minutes), then non-adherent cells are removed bygentle washing. Adhered cells are quantitated by conventional methods(e.g., by staining with crystal violet, lysing the cells, anddetermining the optical density of the lysate). Control wells are coatedwith a known adhesive protein, such as fibronectin or vitronectin.

[0099] The activity of zalpha33 proteins can be measured with asilicon-based biosensor microphysiometer that measures the extracellularacidification rate or proton excretion associated with receptor bindingand subsequent physiologic cellular responses. An exemplary such deviceis the Cytosensor™ Microphysiometer manufactured by Molecular Devices,Sunnyvale, Calif. A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method. See, for example, McConnell et al., Science 257:1906-1912,1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli etal., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including zalpha33 proteins, their agonists, and antagonists.The microphysiometer can be used to measure responses of azalpha33-responsive eukaryotic cell, compared to a control eukaryoticcell that does not respond to zalpha33 polypeptide. Zalpha33-responsiveeukaryotic cells comprise cells into which a receptor for zalpha33 hasbeen transfected creating a cell that is responsive to zalpha33, as wellas cells naturally responsive to zalpha33 such as cells derived fromvascular tissue. Differences, measured by a change in extracellularacidification, in the response of cells exposed to zalpha33 polypeptiderelative to a control not exposed to zalpha33, are a direct measurementof zalpha33-modulated cellular responses. Moreover, suchzalpha33-modulated responses can be assayed under a variety of stimuli.The present invention thus provides methods of identifying agonists andantagonists of zalpha33 proteins, comprising providing cells responsiveto a zalpha33 polypeptide, culturing a first portion of the cells in theabsence of a test compound, culturing a second portion of the cells inthe presence of a test compound, and detecting a change in a cellularresponse of the second portion of the cells as compared to the firstportion of the cells. The change in cellular response is shown as ameasurable change in extracellular acidification rate. Culturing a thirdportion of the cells in the presence of a zalpha33 protein and theabsence of a test compound provides a positive control for thezalpha33-responsive cells and a control to compare the agonist activityof a test compound with that of the zalpha33 polypeptide. Antagonists ofzalpha33 can be identified by exposing the cells to zalpha33 protein inthe presence and absence of the test compound, whereby a reduction inzalpha33-stimulated activity is indicative of antagonist activity in thetest compound.

[0100] Expression of zalpha33 polynucleotides in animals provides modelsfor further study of the biological effects of overproduction orinhibition of protein activity in vivo. Zalpha33-encodingpolynucleotides and antisense polynucleotides can be introduced intotest animals, such as mice, using viral vectors or naked DNA, ortransgenic animals can be produced.

[0101] One in vivo approach for assaying proteins of the presentinvention utilizes viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, retroviruses, vaccinia virus,and adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection.

[0102] By deleting portions of the adenovirus genonle, larger inserts(up to 7 kb) of heterologous DNA can be accommodated. These inserts canbe incorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential El gene is deleted from the viral vector, and the virus willnot replicate unless the El gene is provided by the host cell (e.g., thehuman 293 cell line). When intravenously administered to intact animals,adenovirus primarily targets the liver. If the adenoviral deliverysystem has an E1 gene deletion, the virus cannot replicate in the hostcells. However, the host's tissue (e.g., liver) will express and process(and, if a signal sequence is present, secrete) the heterologousprotein. Secreted proteins will enter the circulation in the highlyvascularized liver, and effects on the infected animal can bedetermined.

[0103] An alternative method of gene delivery comprises removing cellsfrom the body and introducing a vector into the cells as a naked DNAplasmid. The transformed cells are then re-implanted in the body. NakedDNA vectors are introduced into host cells by methods known in the art,including transfection, electroporation, microinjection, transduction,cell fusion, DEAE dextran, calcium phosphate precipitation, use of agene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol.Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967,1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.

[0104] Transgenic mice, engineered to express a zalpha33 gene, and micethat exhibit a complete absence of zalpha33 gene function, referred toas “knockout mice” (Snouwaert et al., Science 257:1083, 1992), can alsobe generated (Lowell et al., Nature 366:740-742, 1993). These mice canbe employed to study the zalpha33 gene and the protein encoded therebyin an in vivo system. Transgenic mice are particularly useful forinvestigating the role of zalpha33 proteins in early development in thatthey allow the identification of developmental abnormalities or blocksresulting from the over- or underexpression of a specific factor. Seealso, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science277:48-50, 1997. Suitable promoters for transgenic expression includepromoters from metallothionein and albumin genes.

[0105] Antisense methodology can be used to inhibit zalpha33 genetranscription to examine the effects of such inhibition in vivo.Polynucleotides that are complementary to a segment of azalpha33-encoding polynucleotide (e.g., a polynucleotide as set forth inSEQ ID NO:1) are designed to bind to zalpha33-encoding mRNA and toinhibit translation of such mRNA. Such antisense oligonucleotides canalso be used to inhibit expression of zalpha33 polypeptide-encodinggenes in cell culture.

[0106] Most four-helix bundle cytokines as well as other proteinsproduced by activated lymphocytes play an important biological role incell differentiation, activation, recruitment and homeostasis of cellsthroughout the body. Zalpha33 and inhibitors of zalpha33 activity areexpected to have a variety of therapeutic applications. Thesetherapeutic applications include treatment of diseases which requireimmune regulation, including autoimmune diseases such as rheumatoidarthritis, multiple sclerosis, myasthenia gravis, systemic lupuserythematosis, and diabetes. Zalpha33 may be important in the regulationof inflammation, and therefore would be useful in treating rheumatoidarthritis, asthma and sepsis. There may be a role of zalpha33 inmediating tumorigenesis, whereby a zalpha33 antagonist would be usefulin the treatment of cancer. Zalpha33 may be useful in modulating theimmune system, whereby zalpha33 and zalpha33 antagonists may be used forreducing graft rejection, preventing graft-vs-host disease, boostingimmunity to infectious diseases, treating immunocompromised patients(e.g., HIV⁺ patients), or in improving vaccines.

[0107] Zalpha33 polypeptides can be administered alone or in combinationwith other vasculogenic or angiogenic agents, including VEGF. When usingzalpha33 in combination with an additional agent, the two compounds canbe administered simultaneously or sequentially as appropriate for thespecific condition being treated.

[0108] For pharmaceutical use, zalpha33 proteins are formulated fortopical or parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. In general, pharmaceuticalformulations will include a zalpha33 polypeptide in combination with apharmaceutically acceptable vehicle, such as saline, buffered saline, 5%dextrose in water, or the like. Formulations may further include one ormore excipients, preservatives, solubilizers, buffering agents, albuminto prevent protein loss on vial surfaces, etc. Methods of formulationare well known in the art and are disclosed, for example, in Remington:The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Zalpha33 will usually be used in aconcentration of about 10 to 100 μg/ml of total volume, althoughconcentrations in the range of 1 ng/ml to 1000 μg/ml may be used. Fortopical application, such as for the promotion of wound healing, theprotein will be applied in the range of 0.1-10 μg/cm² of wound area,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. Dosing is daily or intermittentlyover the period of treatment. Intravenous administration will be bybolus injection or infusion over a typical period of one to severalhours. Sustained release formulations can also be employed. In general,a therapeutically effective amount of zalpha33 is an amount sufficientto produce a clinically significant change in the treated condition,such as a clinically significant change in hematopoietic or immunefunction, a significant reduction in morbidity, or a significantlyincreased histological score.

[0109] Zalpha33 proteins, agonists, and antagonists are useful formodulating the expansion, proliferation, activation, differentiation,migration, or metabolism of responsive cell types, which include bothprimary cells and cultured cell lines. Of particular interest in thisregard are hematopoietic cells (including stem cells and mature myeloidand lymphoid cells), endothelial cells, smooth muscle cells,fibroblasts, and hepatocytes. Zalpha33 polypeptides are added to tissueculture media for these cell types at a concentration of about 10 pg/mlto about 100 ng/ml. Those skilled in the art will recognize thatzalpha33 proteins can be advantageously combined with other growthfactors in culture media.

[0110] Within the laboratory research field, zalpha33 proteins can alsobe used as molecular weight standards or as reagents in assays fordetermining circulating levels of the protein, such as in the diagnosisof disorders characterized by over- or under-production of zalpha33protein or in the analysis of cell phenotype.

[0111] Zalpha33 proteins can also be used to identify inhibitors oftheir activity. Test compounds are added to the assays disclosed aboveto identify compounds that inhibit the activity of zalpha33 protein. Inaddition to those assays disclosed above, samples can be tested forinhibition of zalpha33 activity within a variety of assays designed tomeasure receptor binding or the stimulation/inhibition ofzalpha33-dependent cellular responses. For example, zalpha33-responsivecell lines can be transfected with a reporter gene construct that isresponsive to a zalpha33-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a zalpha33-activated serum response element (SRE) operablylinked to a gene encoding an assayable protein, such as luciferase.Candidate compounds, solutions, mixtures or extracts are tested for theability to inhibit the activity of zalpha33 on the target cells asevidenced by a decrease in zalpha33 stimulation of reporter geneexpression. Assays of this type will detect compounds that directlyblock zalpha33 binding to cell-surface receptors, as well as compoundsthat block processes in the cellular pathway subsequent toreceptor-ligand binding. In the alternative, compounds or other samplescan be tested for direct blocking of zalpha33 binding to receptor usingzalpha33 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradishperoxidase, FITC, and the like). Within assays of this type, the abilityof a test sample to inhibit the binding of labeled zalpha33 to thereceptor is indicative of inhibitory activity, which can be confirmedthrough secondary assays. Receptors used within binding assays may becellular receptors or isolated, immobilized receptors.

[0112] As used herein, the term “antibodies” includes polyclonalantibodies, monoclonal antibodies, antigen-binding fragments thereofsuch as F(ab′)₂ and Fab fragments, single chain antibodies, and thelike, including genetically engineered antibodies. Non-human antibodiesmay be humanized by grafting non-human CDRs onto human framework andconstant regions, or by incorporating the entire non-human variabledomains (optionally “cloaking” them with a human-like surface byreplacement of exposed residues, wherein the result is a “veneered”antibody). In some instances, humanized antibodies may retain non-humanresidues within the human variable region framework domains to enhanceproper binding characteristics. Through humanizing antibodies,biological half-life may be increased, and the potential for adverseimmune reactions upon administration to humans is reduced. One skilledin the art can generate humanized antibodies with specific and differentconstant domains (i.e., different Ig subclasses) to facilitate orinhibit various immune functions associated with particular antibodyconstant domains. Antibodies are defined to be specifically binding ifthey bind to a zalpha33 polypeptide or protein with an affinity at least10-fold greater than the binding affinity to control (non-zalpha33)polypeptide or protein. The affinity of a monoclonal antibody can bereadily determined by one of ordinary skill in the art (see, forexample, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

[0113] Methods for preparing polyclonal and monoclonal antibodies arewell known in the art (see for example, Hurrell, J. G. R., Ed.,Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,Inc., Boca Raton, Fla., 1982, which is incorporated herein byreference). As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats. The immunogenicity of a zalpha33 polypeptide may beincreased through the use of an adjuvant such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of a zalpha33 polypeptide or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is “hapten-like”, such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

[0114] Alternative techniques for generating or selecting antibodiesinclude in vitro exposure of lymphocytes to zalpha33 polypeptides, andselection of antibody display libraries in phage or similar vectors(e.g., through the use of immobilized or labeled zalpha33 polypeptide).Human antibodies can be produced in transgenic, non-human animals thathave been engineered to contain human immunoglobulin genes as disclosedin WIPO Publication WO 98/24893. It is preferred that the endogenousimmunoglobulin genes in these animals be inactivated or eliminated, suchas by homologous recombination.

[0115] A variety of assays known to those skilled in the art can beutilized to detect antibodies which specifically bind to zalpha33polypeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations,enzyme-linked immunosorbent assays (ELISA), dot blot assays, Westernblot assays, inhibition or competition assays, and sandwich assays.

[0116] Antibodies to zalpha33 may be used for affinity purification ofthe protein, within diagnostic assays for determining circulating levelsof the protein; for detecting or quantitating soluble zalpha33polypeptide as a marker of underlying pathology or disease; forimmunolocalization within whole animals or tissue sections, includingimmunodiagnostic applications; for immunohistochernistry; and asantagonists to block protein activity in vitro and in vivo. Antibodiesto zalpha33 may also be used for tagging cells that express zalpha33;for affinity purification of zalpha33 polypeptides and proteins; inanalytical methods employing FACS; for screening expression libraries;and for generating anti-idiotypic antibodies. Antibodies can be linkedto other compounds, including therapeutic and diagnostic agents, usingknown methods to provide for targetting of those compounds to cellsexpressing receptors for zalpha33. For certain applications, includingin vitro and in vivo diagnostic uses, it is advantageous to employlabeled antibodies. Suitable direct tags or labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmarkers, chemiluminescent markers, magnetic particles and the like;indirect tags or labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. Antibodies of thepresent invention may also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications (e.g., inhibition of cellproliferation). See, in general, Ramakrishnan et al., Cancer Res.56:1324-1330, 1996.

[0117] Polypeptides and proteins of the present invention can be used toidentify and isolate receptors. Zalpha33 receptors may be involved ingrowth regulation in the liver, blood vessel formation, and otherdevelopmental processes. For example, zalpha33 proteins and polypeptidescan be immobilized on a column, and membrane preparations run over thecolumn (as generally disclosed in Immobilized Affinity LigandTechniques, Hermanson et al., eds., Academic Press, San Diego, Calif.,1992, pp.195-202). Proteins and polypeptides can also be radiolabeled(Methods Enzymol., vol. 182, “Guide to Protein Purification”, M.Deutscher, ed., Academic Press, San Diego, 1990, 721-737) orphotoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514,1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and usedto tag specific cell-surface proteins. In a similar manner, radiolabeledzalpha33 proteins and polypeptides can be used to clone the cognatereceptor in binding assays using cells transfected with an expressioncDNA library.

[0118] The present invention also provides reagents for use indiagnostic applications. For example, the zalpha33 gene, a probecomprising zalpha33 DNA or RNA, or a subsequence thereof can be used todetermine the presence of mutations at or near the zalpha33 locus.Detectable chromosomal aberrations at the zalpha33 gene locus include,but are not limited to, aneuploidy, gene copy number changes,insertions, deletions, restriction site changes, and rearrangements.These aberrations can occur within the coding sequence, within introns,or within flanking sequences, including upstream promoter and regulatoryregions, and may be manifested as physical alterations within a codingsequence or changes in gene expression level. Analytical probes willgenerally be at least 20 nucleotides in length, although somewhatshorter probes (14-17 nucleotides) can be used. PCR primers are at least5 nucleotides in length, often 15 or more nt, commonly 20-30 nt. Shortpolynucleotides can be used when a small region of the gene is targettedfor analysis. For gross analysis of genes, a polynucleotide probe maycomprise an entire exon or more. Probes will generally comprise apolynucleotide linked to a signal-generating moiety such as aradionucleotide. In general, these diagnostic methods comprise the stepsof (a) obtaining a genetic sample from a patient; (b) incubating thegenetic sample with a polynucleotide probe or primer as disclosed above,under conditions wherein the polynucleotide will hybridize tocomplementary polynucleotide sequence, to produce a first reactionproduct; and (c) comparing the first reaction product to a controlreaction product. A difference between the first reaction product andthe control reaction product is indicative of a genetic abnormality inthe patient. Genetic samples for use within the present inventioninclude genomic DNA, cDNA, and RNA. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO: 1, thecomplement of SEQ ID NO: 1, or an RNA equivalent thereof. Suitable assaymethods in this regard include molecular genetic techniques known tothose in the art, such as restriction fragment length polymorphism(RFLP) analysis, short tandem repeat (STR) analysis employing PCRtechniques, ligation chain reaction (Barany, PCR Methods andApplications 1:5-16, 1991), ribonuclease protection assays, and othergenetic linkage analysis techniques known in the art (Sambrook et al.,ibid.; Ausubel et. al., ibid.; A. J. Marian, Chest 108:255-65, 1995).Ribonuclease protection assays (see, e.g., Ausubel et al., ibid., ch. 4)comprise the hybridization of an RNA probe to a patient RNA sample,after which the reaction product (RNA-RNA hybrid) is exposed to RNase.Hybridized regions of the RNA are protected from digestion. Within PCRassays, a patient genetic sample is incubated with a pair ofpolynucleotide primers, and the region between the primers is amplifiedand recovered. Changes in size or amount of recovered product areindicative of mutations in the patient. Another PCR-based technique thatcan be employed is single strand conformational polymorphism (SSCP)analysis (Hayashi, PCR Methods and Applications 1:34-38, 1991).

[0119] Radiation hybrid mapping is a somatic cell genetic techniquedeveloped for constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245-50, 1990). Partial or fullknowledge of a gene's sequence allows one to design PCR primers suitablefor use with chromosomal radiation hybrid mapping panels. Radiationhybrid mapping panels that cover the entire human genome arecommercially available, such as the Stanford G3 RH Panel and theGeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, Ala.). Thesepanels enable rapids PCR-based chromosomal localizations and ordering ofgenes, sequence-tagged sites (STSs), and other nonpolymorphic andpolymorphic markers within a region of interest, and the establishmentof directly proportional physical distances between newly discoveredgenes of interest and previously mapped markers. The precise knowledgeof a gene's position can be useful for a number of purposes,including: 1) determining if a sequence is part of an existing contigand obtaining additional surrounding genetic sequences in various forms,such as YACs, BACs or cDNA clones; 2) providing a possible candidategene for an inheritable disease which shows linkage to the samechromosomal region; and 3) cross-referencing model organisms, such asmouse, which may aid in determining what function a particular genemight have.

[0120] Sequence tagged sites (STSs) can also be used independently forchromosomal localization. An STS is a DNA sequence that is unique in thehuman genome and can be used as a reference point for a particularchromosome or region of a chromosome. An STS is defined by a pair ofoligonucleotide primers that are used in a polymerase chain reaction tospecifically detect this site in the presence of all other genomicsequences. Since STSs are based solely on DNA sequence they can becompletely described within an electronic database, for example,Database of Sequence Tagged Sites (dbSTS), GenBank (National Center forBiological Information, National Institutes of Health, Bethesda, Md.http://www.ncbi.nlm.riih.gov), and can be searched with a gene sequenceof interest for the mapping data contained within these short genomiclandmark STS sequences.

[0121] The polypeptides, nucleic acid and/or antibodies of the presentinvention may be used in diagnosis or treatment of disorders associatedwith cell loss or abnormal cell proliferation (including cancer).Labeled zalpha33 polypeptides may be used for imaging tumors or othersites of abnormal cell proliferation.

[0122] Inhibitors of zalpha33 activity (zalpha33 antagonists) includeanti-zalpha33 antibodies and soluble zalpha33 receptors, as well asother peptidic and non-peptidic agents (including ribozymes). Suchantagonists can be used to block the effects of zalpha33 on cells ortissues. Of particular interest is the use of antagonists of zalpha33activity in cancer therapy. As early detection methods improve itbecomes possible to intervene at earlier times in tumor development,making it feasible to use inhibitors of growth factors to block cellproliferation, angiogenesis, and other events that lead to tumordevelopment and metastasis. Inhibitors are also expected to be useful inadjunct therapy after surgery to prevent the growth of residual cancercells. Inhibitors can also be used in combination with other cancertherapeutic agents.

[0123] In addition to antibodies, zalpha33 inhibitors include smallmolecule inhibitors and inactive receptor-binding fragments of zalpha33polypeptides. Inhibitors are formulated for pharmaceutical use asgenerally disclosed above, taking into account the precise chemical andphysical nature of the inhibitor and the condition to be treated. Therelevant determinations are within the level of ordinary skill in theformulation art.

[0124] Polynucleotides encoding zalpha33 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzalpha33 activity. If a mammal has a mutated or absent zalpha33 gene, azalpha33 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zalpha33 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are commonly used. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-330, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-630,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-3101, 1987; Samulski et al., J. Virol. 63:3822-3888,1989). Within another embodiment, a zalpha33 gene can be introduced in aretroviral vector as described, for example, by Anderson et al., U.S.Pat. No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S.Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz etal., J. Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;Dougherty et al., WIPO Publication WO 95/07358; and Kuo et al., Blood82:845, 1993. Alternatively, the vector can be introduced byliposome-mediated transfection (“lipofection”). Synthetic cationiclipids can be used to prepare liposomes for in vivo transfection of agene encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA85:8027-8031, 1988). The use of lipofection to introduce exogenous genesinto specific organs in vivo has certain practical advantages, includingmolecular targeting of liposomes to specific cells. Directingtransfection to particular cell types is particularly advantageous in atissue with cellular heterogeneity, such as the pancreas, liver, kidney,and brain. Lipids may be chemically coupled to other molecules for thepurpose of targeting. Peptidic and non-peptidic molecules can be coupledto liposomes chemically. Within another embodiment, cells are removedfrom the body, a vector is introduced into the cells as a naked DNAplasmid, and the transformed cells are re-implanted into the body asdisclosed above.

[0125] Antisense methodology can be used to inhibit zalpha33 genetranscription in a patient as generally disclosed above.

[0126] Zalpha33 polypeptides and anti-zalpha33 antibodies can bedirectly or indirectly conjugated to drugs, toxins, radionuclides andthe like, and these conjugates used for in vivo diagnostic ortherapeutic applications. For instance, polypeptides or antibodies ofthe present invention may be used to identify or treat tissues or organsthat express a corresponding anti-complementary molecule (receptor orantigen, respectively, for instance). More specifically, zalpha33polypeptides or anti-zalpha33 antibodies, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues, or organs that expressthe anti-complementary molecule.

[0127] Suitable detectable molecules can be directly or indirectlyattached to the polypeptide or antibody, and include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles, and the like. Suitablecytotoxic molecules can be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporin,and the like), as well as therapeutic radionuclides, such as iddine-131,rhenium-188 or yttrium-90. These can be either directly attached to thepolypeptide or antibody, or indirectly attached according to knownmethods, such as through a chelating moiety. Polypeptides or antibodiescan also be conjugated to cytotoxic drugs, such as adriamycin. Forindirect attachment of a detectable or cytotoxic molecule, thedetectable or cytotoxic molecule may be conjugated with a member of acomplementary/anticomplementary pair, where the other member is bound tothe polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

[0128] Polypeptide-toxin fusion proteins or antibody/fragment-toxinfusion proteins may be used for targeted cell or tissue inhibition orablation, such as in cancer therapy. Of particular interest in thisregard are conjugates of a zalpha33 polypeptide and a cytotoxin, whichcan be used to target the cytotoxin to a tumor or other tissue that isundergoing undesired angiogenesis or neovascularization. Target cells(i.e., those displaying the zalpha33 receptor) bind the zalpha33-toxinconjugate, which is then internalized, killing the cell. The effects ofreceptor-specific cell killing (target ablation) are revealed by changesin whole animal physiology or through histological examination. Thus,ligand-dependent, receptor-directed cyotoxicity can be used to enhanceunderstanding of the physiological significance of a protein ligand. Anexemplary toxin is saporin. Mammalian cells have no receptor forsaporin, which is non-toxic when it remains extracellular.

[0129] In another embodiment, zalpha33-cytokine fusion proteins orantibody/fragment-cytokine fusion proteins may be used for enhancing invitro cytotoxicity (for instance, that mediated by monoclonal antibodiesagainst tumor targets) and for enhancing in vivo killing of targettissues (for example, blood and bone marrow cancers). See, generally,Hornick et al., Blood 89:4437-4447, 1997). In general, cytokines aretoxic if administered systemically. The described fusion proteins enabletargeting of a cytokine to a desired site of action, such as a cellhaving binding sites for zalpha33, thereby providing an elevated localconcentration of cytokine. Suitable cytokines for this purpose include,for example, interleukin-2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF). Such fusion proteins may be used to causecytokine-induced killing of tumors and other tissues undergoingangiogenesis or neovascularization.

[0130] The bioactive polypeptide or antibody conjugates described hereincan be delivered intravenously, intra-arterially or intraductally, ormay be introduced locally at the intended site of action.

[0131] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLE 1

[0132] Analysis of tissue distribution was performed by the Northernblotting technique using commercially available Blots of human RNA(Human Multiple Tissue Northern Blots I, II, and m; and Master DotBlots; Clontech Laboratories, Inc., Palo Alto, Calif.). A probe wasobtained by restriction digest of a human zalpha33 clone with EcoRI andPvuII, resulting in two cDNA fragments of 288 bp and 326 bp. Thereaction mixture was electrophoresed on a 2% agarose gel, and thecorresponding bands were excised and purified using commerciallyavailable gel purification reagents and protocol (QIAEX® II gelextraction kit; Qiagen, Valencia, Calif.). The purified DNA wasradioactively labeled with P using a commercially available kit(Rediprime™ II random-prime labeling system; Amersham Corp., ArlingtonHeights, Ill.). The probe was purified using a commercially availablesize exclusion column (NucTrap® column; Stratagene, La Jolla, Calif.). Acommercially available hybridization solution (ExpressHyb™ HybridizationSolution; Clontech Laboratories Inc., Palo Alto, Calif.) was used forhybridization and prehybridization. The final hybridization solutioncontained 8 ml ExpressHyb™ solution, 80 μl sheared salmon sperm DNA (10mg/ml; obtained from 5 Prime-3 Prime, Boulder, Colo.) and 1.6×10⁷ cpmlabeled probe. Hybridization took place overnight at 55° C. Afterhybridization the blots were washed in 2× SSC, 0.1% SDS at roomtemperature; then in 2× SSC, 0.1% SDS at 60° C. followed by a 0.1× SSC,0.1% SDS wash at 60° C. The blots were exposed to film (Biomax AR film,Eastman Kodak Co., Rochester N.Y.) for three days and developed.

[0133] One major transcript size was observed on the Human MultipleTissue blots at ˜1.0 kb in all tissues. The signal was strongest inheart, liver, pancreas, testis, ovary, and thyroid tissue, with othertissues showing moderately lower expression levels. Signals on theMaster Dot blot were present in all tisues with slightly higher signalsin aorta, testis, pituitary gland, thyroid gland, mamary gland, thymus,lung, fetal thymus, and fetal lung. Expression was also seen in blots ofTHP1 and U937 monocyte cell lines.

EXAMPLE 2

[0134] An expression plasmid encoding full-length mouse zalpha33 wasconstructed using the expression vector pEZE2. The vector pEZE2 wasderived from pDC312 by the addition of additional restriction enzymerecognition sites to the multiple cloning site. pDC312 and pEZE2 containan EASE segment, as described in WO 97/25420, which can improveexpression of recombinant proteins two- to eight-fold in mammalian cells(e.g., Chinese Hamster Ovary (CHO) cells). The pEZE2 expression unitcontains the CMV enhancer/promoter, the adenovirus tripartite leadersequence, a multiple cloning site for insertion of the coding region forthe recombinant protein, an encephalomyocarditis virus internal ribosomeentry site, a coding segment for mouse dihydrofolate reductase, and theSV40 transcription terminator. In addition, pEZE2 contains an E. coliorigin of replication and a bacterial beta-lactamase gene.

[0135] A DNA fragment encoding zalpha33 with a C-terminal Glu-Glu tag(zalpha33CEE) was generated by PCR using a commercially available kit(AdvantageD 2 PCR Kit, Clontech, Palo Alto, Calif.). The fragmentincluded 5′FseI and 3′ AscI sites for direct cloning into the expressionvector. The 5′ primer contained an FseI site, a Kozak sequence, and thefirst 31 base pairs of the native leader sequence for zalpha33.

[0136] The 3′ primer contained the last 18 base pairs of zalpha33, aGlu-Glu tag sequence, a stop codon, and an AscI site. The PCR mixtureincluded lul of template (plasmid containing the full-length mousezalpha33 sequence). The reaction was run at 94° C., 1 minute; then 25cycles of 94° C., 30 seconds; 55° C., 30 seconds; 68° C., 1 minute; thena final extension at 72° C. for 7 minutes. The PCR-generated fragmentwas purified using a commercially available kit (Qiaquick™ PCRPurification Kit, Qiagen Inc., Valencia, Calif.) and digested withrestriction enzymes AscI and FseI (New England Biolabs, Beverly, Mass.)in a single, 100-μl reaction. Five micrograms of the expression vectorpEZE2 were also digested with FseI and AscI in a single, 100-μlreaction. The digested DNA was fractionated by agarose gelelectrophoresis, and the DNA fragments were isolated and purified usinga commercially available kit (Qiaquick™ Gel Extraction Kit, QiagenInc.).

[0137] Five microliters of the zalpha33CEE DNA fragment and 1 ul of thepEZE2 vector fragment were ligated overnight at room temperature usingT4 DNA ligase (high concentration) and reaction buffer obtained from NewEngland Biolabs, Beverly, Mass. One microliter of the ligation mixturewas added to 25 μl of electrocompetant E. coli strain DH10B (LifeTechnologies) in a 0.2-cm cuvette. The mixture was electroporated(BioRad E. coli Pulser) at 2.3 kv. To the cuvette, 1 mL of LB broth wasadded, and 100 μl of the mix was plated onto LB/Ampicillin agar plates.The plates were incubated overnight at 37° C., and 16 isolated colonieswere picked for DNA mini prep using a commercially available kit(obtained from Qiagen Inc.). Individual clones were screened by PCR forthe presence of zalpha33CEE DNA, using the above-mentioned primers. DNAsequencing was performed on clones #1-6, to verify the correctfull-length sequence. One clone contained the correct expected sequence,from which DNA was prepared using a commercially available kit (Qiagen®Plasmid Maxi Kit, Qiagen Inc.). The plasmid was designated pKFO248.

EXAMPLE 3

[0138] Plasmid pKFO248 was prepared for transfection into Chinesehamster ovary (CHO) cells. To insure sterility a single ethanolprecipitation step was performed by combining 200 μg of plasmid DNA with20 μl of 10 mg/ml sheared salmon sperm carrier DNA (5′ <3′ Inc. Boulder,Colo.), 22 μl of 3M sodium acetate (pH 5;2), and 484 μl of 100% ethanol(Gold Shield Chemical Co., Hayward, Calif.) and incubating the mixtureon ice for 25 minutes. After incubation, the mixture was spun at 14,000RPM in a microcentrifuge at 4° C., the supernatant was removed, and thepellet was washed twice with 0.5 ml 70% ethanol and allowed to air dryuntil it appeared as an opaque white color.

[0139] Protein-free and serum-free suspension-adapted CHO DG44 cells(Chasin et al., Som. Cell Molec. Genet. 12:555-666, 1986) were takenfrom a frozen stock (passage 36) and preparerd for transfection byculturing in PFCHO media (JRH Biosciences, Lenexa, Kans.), 4 mML-Glutamine, (JRH Biosciences), and 1× HT Supplement (Life TechnologiesLot # 1024782) at 37° C. and 5% CO₂ in shake flasks at 120 RPM on arotating shaker platform. The cells were allowed to recover from theprocess of thaw before being transfected at passage 38 with the plasmidzalpha 33 Mouse-CEE/pEZE2 (PKFO 248 linearized with Fsp) by High CopyElectroporation.

[0140] The CHO DG44 cells were transfected by electroporation. While theDNA pellet was drying, 10e6 total cells (16.5ml) were spun in a 25 mlconical centrifuge tube at 900 RPM for 5 minutes. The cells wereresuspended into a total volume of 300 μl in PFCHO media as above andplaced in an electroporation cuvette with a 0.4 cm electrode gap (GenePulser® Cuvette; Bio-Rad Laboratories, Inc., Hercules, Calif.). Afterapproximately 50 minutes of drying time the plasmid DNA was resuspendedinto 500 μl of PFCHO growth media and added to the cuvette so that thetotal volume did not exceed 800 microliters. The mixture was allowed tosit at room temperature for 5 minutes to decrease bubble formation. Thecuvette was placed in an electroporator (Gene Pulser® II; Bio-RadLaboratories, Inc.) set at 0.296 kV and 0.950 HC (high capacitence) andelectroporated immediately with an actual reading of 0.310 kV and a timeconstant of 15.7 milliseconds.

[0141] The cells were allowed to sit 5 minutes at room temperature torecover before placement in 20 ml total volume of PFCHO media in atissue culture flask using a sterile glass 9-inch transfer pipetpreviously baked for 4 hours at 260° C. The flask was placed at 37° C.and 5% CO₂ for 48 hours, at which time the cells were counted byhemocytometer utilizing trypan blue exclusion and put into PFCHOselection media without HT supplement and containing 200 mM methotrexate(Cal Biochem; San Diego, Calif.).

[0142] Upon recovery from the selection process conditioned mediacontaining the secreted zalpha 33 protein is assayed by Western Blot.Neat 72-hour conditioned media is diluted 1:2 in reducing sample buffer(NuPAGE™ 2× buffer; Novex, San Diego, Calif.) containing 100 mM DTT (ICNBiochemicals, Costa Mesa, Calif.), and 25-μl samples are loaded intoappropriate wells of a 1.0 mM×12 well, 4-12% Bis-Tris Gel (NuPAGE™;Novex) and run at 150 volts for approximateley one hour. The gel is runwith a protein standard (Glu-Glu tagged leptin produced in Pichiamethanolica) is serially diluted ranging from 100 ng down to 6.25 ngtotal per lane and 10 μl of commercially available molecular weightmarkers (SeeBlue™; Novex). Proteins are transferred to a 0.2 μmnitrocellulose membrane (Novex) at 30 volts for approximately one hour.The blot is blocked in 10% nonfat dry milk (Carnation) in Western Abuffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA,0.05% octylphenylpolyethylene glycol (Igepal®-CA630)) either overnightat 4° C. or for one hour at room temperature on a rotating shakerplatform. Thirty ml of mouse anti-Glu-Glu monoclonal antibody (BabCOBerkeley Antibody Company, Richmond, Calif.) diluted to 0.1 μg/ml in2.5% nonfat dry milk in Western A is overlayed on the membrane, and theblot is incubated at room temperature on a rotating platform for onehour. The antibody solution is then discarded, and the membrane isrinsed three times (about 50 ml each time) with Western A. The blot isthen incubated with 30 ml of goat anti-mouse IgG-HRP secondary antibody(1 mg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) at a1:2000 dilution under the same conditions for one hour. The secondaryantibody solution is discarded and the membrane rinsed as describedabove. protein is detected by chemiluminescence using commerciallyavailable reagents (ECL™ Plus Western Blotting Detection System;Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) and instrumentation(Lumi-Imager™ F1; Roche Molecular Biochemicals, Indianapolis, Ind.).

EXAMPLE 4

[0143] The protein coding region of mouse zalpha33 was amplified by PCRusing primers that added FseI and Asci restriction sites at the 5′ and3′ termini respectively. PCR primers ZC23310 (SEQ ID NO:8) and ZC23311(SEQ ID NO:9) were used with template plasmid containing the full-lengthmouse zalpha33 cDNA in a PCR reaction as follows: one cycle at 95° C.for 5 minutes; followed by 15 cycles at 95° C. for 0.5 min., 58° C. for0.5 min., and 72° C. for 0.5 min.; followed by 72° C. for 10 min.;followed by a 4° C. soak. The PCR reaction product was loaded onto a1.2% (low melt) agarose (SeaPlaque(D GTG; FMC Corp, Rockland, Me.) gelin TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). The zalpha33 PCRproduct was excised from the gel. The gel slice was melted at 65° C.,extracted twice with an equal volume of Tris-buffered phenol, and EtOHprecipitated. The DNA was resuspended in 10 μl dH₂O, digested withFseI-AscI, phenol/chloroform extracted, EtOH precipitated, andrehydrated in 20 μl TE (Tris/EDTA pH 8). The 537-bp zalpha33 fragmentwas then ligated into the FseI-AscI sites of a modified pAdTrack CMV (Heet al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998). This constructalso contained the green fluorescent protein (GFP) marker gene. The CMVpromoter driving GFP expression was replaced with the SV40 promoter, andthe SV40 polyadenylation signal was replaced with the human growthhormone polyadenylation signal. In addition, the native polylinker wasreplaced with FseI, EcoRV, and AscI sites. This modified form ofpAdTrack CMV was named pZyTrack. Ligation was performed using a DNAligation and screening kit (Fast-Link™; Epicentre Technologies, Madison,Wis.). Clones containing the zalpha33 cDNA were identified by standardminiprep procedures. To linearize the plasmid, approximately 5 μg of thepZyTrack zalpha33 plasmid was digested with PmeI. Approximately 1 μg ofthe linearized plasmid was cotransformed with 200 ng of supercoiledpAdEasy (He et al., ibid.) into BJ5183 cells using an electroporator(Gene Pulser®; Bio-Rad Laboratories, Inc.) set at 2.5 kV, 200 ohms, and25 μFa. The entire co-transformation was plated on 4 LB platescontaining 25 μg/ml kanamycin. The smallest colonies were picked andexpanded in LB/kanamycin, and recombinant adenovirus DNA was identifiedby standard DNA miniprep procedures. Digestion of the recombinantadenovirus DNA with FseI-AscI confirmed the presence of the zalpha33sequence. The recombinant adenovirus miniprep DNA was transformed intoE. coli strain DH10B™ (Life Technologies, Gaithersburg, Md.) competentcells, and DNA was prepared by anion exchange chromatography using acommercially available plasmid isolation kit (QIAGEN® Plasmid Maxi Kit;Qiagen, Inc., Valencia, Calif.).

[0144] Approximately 5 μg of recombinant adenoviral DNA was digestedwith PacI enzyme (New England Biolabs) for 3 hours at 37° C. in areaction volume of 100 μl containing 20-30U of PacI. The digested DNAwas extracted twice with an equal volume of phenol/chloroform andprecipitated with ethanol. The DNA pellet was resuspended in 5 μldistilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies,Inc., Montreal, Canada), inoculated the day before and grown to 60-70%confluence, was transfected with the PacI-digested DNA. ThePacI-digested DNA was diluted up to a total volume of 50 μl with sterileHBS (150 mM NaCl, 20 mM HEPES). In a separate tube, 25 μl of 1 mg/mlN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts (DOTAP)(Boehringer Mannheim, Indianapolis, Ind.) was diluted to a total volumeof 100 μl with HBS. The DNA was added to the DOTAP, mixed gently bypipeting up and down, and left at room temperature for 15 minutes. Themedia was removed from the 293A cells, and the cells were washed with Sml serum-free MEMalpha containing 1 mM sodium pyruvate, 0.1 mM MEMnon-essential amino acids, and 25 mM HEPES buffer (media componentsobtained from Life Technologies, Gaithersburg, Md.). 5 ml of serum-freeMEM was added to the 293A cells and held at 37° C. The DNA/lipid mixturewas added drop-wise to the T25 flask of 293A cells, mixed gently, andincubated at 37° C. for 4 hours. After 4 hours the media containing theDNA/lipid mixture was aspirated off and replaced with 5 ml complete MEMcontaining 5% fetal bovine serum. The transfected cells were monitoredfor GFP expression and formation of foci (viral plaques).

[0145] Seven days after transfection of 293A cells with the recombinantadenoviral DNA, the cells expressed the GFP protein and started to formfoci. The crude viral lysate was collected with a cell scraper andtransferred to a 50-ml conical tube. To release most of the virusparticles from the cells, three freeze/thaw cycles were done in a dryice/ethanol bath and a 37° waterbath.

[0146] The crude lysate was amplified (primary (1°) amplification) toobtain a working “stock” of zalpha33 recombinant adenovirus (rAdV)lysate. Ten 10-cm plates of nearly confluent (80-90%) 293A cells wereset up 20 hours in advance. 200 ml of crude rAdV lysate was added toeach 10-cm plate, and the plates were monitored for 48 to 72 hours forCPE (cytopathic effect) under the white light microscope and expressionof GFP under the fluorescent microscope. When all of the cells showedCPE, the 1° stock lysate was collected, and freeze/thaw cycles wereperformed as described above.

[0147] For secondary (2°) amplification, twenty 15-cm tissue culturedishes of 293A cells were prepared so that the cells were 80-90%confluent. All but 20 ml of 5% MEM media was removed, and each dish wasinoculated with 300-500 ml 10 amplified rAdv lysate. After 48 hours thecells were lysed from virus production. The lysate was collected into250-ml polypropylene centrifuge bottles.

[0148] To purify the rAdV, NP-40 detergent was added to a finalconcentration of 0.5% to the bottles of crude lysate to lyse all cells.Bottles were placed on a rotating platform for 10 minutes, agitating asfast as possible without the bottles falling over. The debris waspelleted by centrifugation at 20,000× G for 15 minutes. The supernatantswere transferred to 250-ml polycarbonate centrifuge bottles, and 0.5volume of 20% PEG-8000/2.5 M NaCl solution was added. The bottles wereshaken overnight on ice. The bottles were centrifuged at 20,000× G for15 minutes, and supernatants were discarded into a bleach solution.Using a sterile cell scraper, the precipitate from 2 bottles wasresuspended in 2.5 ml PBS. The virus solution was placed in 2-mlmicrocentrifuge tubes and centrifuged at 14,000× G for 10 minutes toremove any additional cell debris. The supernatant from the 2-mlmicrocentrifuge tubes was transferred to a 15-ml polypropylene snapcaptube and adjusted to a density of 1.34 g/ml with CsCl. The volume of thevirus solution was estimated, and 0.55 g/ml of CsCl was added. The CsClwas dissolved, and 1 ml of this solution weighed 1.34 g. The solutionwas transferred to polycarbonate thick-walled centrifuge tubes (3.2 ml)(Beckman #362305) and spun at 348,000× G for 3-4 hours at 25° C. in aBeckman Optima LX microultracentrifuge with a TLA-100.4 rotor. The virusformed a white band. Using wide-bore pipette tips, the virus band wascollected.

[0149] The virus preparation was desalted by gel filtration usingcommercially available columns and cross-liked dextran media (PD-10column prepacked with Sephadex®D G-25M; Pharmacia, Piscataway, N.J.).The column was equilibrated with 20 ml of PBS. The virus was loaded andallowed to run into the column. Five ml of PBS was added to the column,and fractions of 8-10 drops were collected. The optical densities of1:50 dilutions of each fraction were determined at 260 nm on aspectrophotometer. A clear absorbance peak was present between fractions7-12. These fractions were pooled, and the optical density (OD) of a1:25 dilution was determined. Virus concentration was calculated by theformula: (OD at 260 nm)(25)(1.1×10¹²)=virions/ml. The OD of a 1:25dilution of the zalpha33 rAdV was 0.059, giving a virus concentration of2.8×1012 virions/nil.

[0150] To store the virus, glycerol was added to the purified virus to afinal concentration of 15%, mixed gently but effectively, and stored inaliquots at −80° C.

[0151] A protocol developed by Quantum Biotechnologies, Inc. (Montreal,Canada) was followed to measure recombinant virus infectivity. Briefly,two 96-well tissue culture plates were seeded with 1×10⁴ 293A cells perwell in MEM containing 2% fetal bovine serum for each recombinant virusto be assayed. After 24 hours, 10-fold dilutions of each virus from1×10-2 to 1×10⁻¹⁴ were made in MEM containing 2% fetal bovine serum. 100μl of each dilution was placed in each of 20 wells. After 5 days at 37°C., wells were read either positive or negative for CPE and a value forplaque forming units/ml (PFU) was calculated.

[0152] TCID₅₀ formulation used was as per Quantum Biotechnologies, Inc.,above. The titer (T) was determined from a plate where virus used wasdiluted from 10⁻² to 10⁻¹⁴, and read 5 days after the infection. At eachdilution a ratio (R) of positive wells for CPE per the total number ofwells was determined.

[0153] To calculate titer of the undiluted virus sample: the factor,“F”=1+d(S−0.5); where “S” is the sum of the ratios (R); and “d” is Log10of the dilution series, for example, “d” is equal to 1 for a ten-folddilution series. The titer of the undiluted sample isT=10^((1+F))=TCID₅₀/ml. To convert TCID₅₀/ml to pfu/ml, 0.7 issubtracted from the exponent in the calculation for titer (T).

[0154] The zalpha33 adenovirus had a titer of 3.2×10¹⁰ pfu/ml.

EXAMPLE 5

[0155] Approximately 10 μg pZytrack vector containing thesequence-confirmed mouse zalpha33 coding region was digested with FseIand AscI. The vector was then ethanol precipitated, and the pellet wasresuspended in Tris-EDTA buffer. The released 537-bp zalpha33 fragmentwas isolated by running the digested vector on a 1.2% agarose gel(SeaPlaque® GTG: FMC Corp., Rockland, Me.) and excising the fragment.DNA was purified using a commercially available kit (QiaQuick™ GelExtraction Kit, Qiagen Inc.).

[0156] The mouse zalpha33 fragment was then ligated into the vectorpTG12-8, which was previously digested with FseI and AscI. The pTG12-8plasmid, designed for expression of a gene of interest in transgenicmice, contains an expression cassette flanked by 10 kb of MT-1 (mousemetallothionein gene) 5′ DNA and 7 kb of MT-1 3′ DNA. The expressioncassette comprises the MT-1 promoter, the rat insulin II intron, apolylinker for the insertion of the desired clone, and the human growthhormone poly A sequence.

[0157] About one microliter of the ligation reaction mixture waselectroporated into E. coli host cells (Electromax DH10B™ cells; LifeTechnologies) according to the supplier's directions, plated onto LBplates containing 100 μg/ml ampicillin, and incubated overnight at 37°C. Colonies were picked and grown in LB media containing 100 μg/mlampicillin. Miniprep DNA was prepared from the picked clones andscreened for the mouse zalpha33 insert by restriction digestion withEcoRi and agarose gel electrophoresis. Maxipreps of the correct pTG12-8murine zalpha33 construct were performed.

[0158] A SalI fragment containing 5′ and 3′ flanking sequences, the MTpromoter, the rat insulin II intron, murine zalpha33 cDNA and the humangrowth hormone poly A sequence was prepared and used for microinjectioninto fertilized mouse oocytes. Two transgenic mice with high zalpha33expression levels had low white blood cell counts.

EXAMPLE 6

[0159] Four confluent T-162 flasks of HaCaT Human keratinocyte cells(Boukamp et al., J. Cell. Biol. 106:761-771, 1988) were trypsinized.Each pellet was resuspended in 4 ml DMEM+5% FBS. The HaCat cells weretransduced by adding purified mouse zalpha33 recombinant adenovirus(AdZy/zalpha33) or parental adenovirus at a MOI of 500 particles percell and shaking slowly at 37° C. for 1 hour. The cells were thentransfered to two T-162 flasks each containing 30 ml growth media andincubated 48 hours (until confluent). The cells were rinsed twice with1× PBS and re-fed with 30 ml per flask serum-freelphenol-red-free DMEMcontaining L-glutamine, sodium pyruvate and HEPES buffer. The cells wereincubated and harvested three times at 72-hour intervals. The collectedconditioned media were centrifuged to remove cellular debris andconcentrated to 10× using Millipore 80 ml, 5K cut-off centrifugal filterdevices. The concentrates were frozen in l-ml aliquots at −80° C.

EXAMPLE 7

[0160] Mouse zalpha33 was assayed in an aortic ring outgrowth assay(Nicosia and Ottinetti, Laboratory Investigation 63(1):115, 1990;Villaschi and Nicosia, Am. J. Pathology 143(l):181-190, 1993). Thoracicaortas were isolated from 1-2 month old SD female rats and transferredto petri dishes containing HANK's buffered salt solution. The aortaswere flushed with additional HANK's buffered salt solution to removeblood, and adventitial tissue surrounding the aortas was carefullyremoved. Cleaned aortas were transferred to petri dishes containingserum-free basal medium (EBM; obtained from Clonetics, San Diego,Calif.). Aortic rings were obtained by slicing approximately 1-mmsections using a scalpel blade. The ends of the aortas used to hold theaorta in place were not used. The rings were rinsed in fresh EBM basalmedia and placed individually in wells of a 24-well plate coated withbasement membrane matrix (Matrigel®; Becton Dickinson, Franklin Lakes,N.J.). The rings were overlayed with an additional 50 μl of Matrigel andplaced at 37° C. for 30 minutes to allow the matrix to gel. Test sampleswere diluted in EBM basal serum-free media supplemented with 100units/ml penicillin, 100 μg/ml streptomycin and HEPES buffer and addedat 1 ml/well. Background control was EBM basal serum-free media alone.Basic FGF (R&D Systems, Minneapolis, Minn.) at 20 ng/ml was used as apositive control. AdZy/zalpha33 adenovirus was added to wells, assuminga cell count of 500,000 cells and a multiplicity of infection of 5000particles/cell. A null pZyTrack adenovirus (zPar) was used as a control.10× conditioned media generated in HaCat keratinocyte cells by a 4 daytranduction with the AdZy/zalpha33 at an MOI of 500 particles per cellwas also used in this assay. A control conditioned media generated usingthe zPar rAdenovirus to transduce HaCat cells was used as the matching.negative control. Samples were added in a minimum of quadruplets. Ringswere incubated for 5-7 days at 37° C. and analyzed for growth. Aorticoutgrowth was scored by multiple, blinded observers using 0 as no growthand 4 as maximum growth. Zalpha33/HaCat conditioned media generated bytransduction with the AdZy/zalpha33 produced a significant increase inoutgrowth as compared to controls, and was comparable to other potentgrowth factors (e.g., bFGF).

EXAMPLE 8

[0161] Various human tissues, including heart, pancreas, testis, liver,ovary and appendix, were isolated and screened for zalpha33 expressionby in situ hybridization. The tissues were fixed overnight in 10%neutral phosphate-buffered formalin (Surgipath, Richmond, Ill.), andembedded in paraffin (Oxford Scientific, St. Louis, Mo.) using standardtechniques. Tissues were sectioned at 4 to 8 microns. Tissues wereprepared using a standard protocol (“Development of non-isotopic in situhybridization” at http://dir.niehs.nih.gov/dirlep/ish.html). Briefly,tissue sections were deparaffinized with a commercially availablehistological clearing agent HistoClear™; National Diagnostics, Atlanta,Ga.) and then dehydrated with ethanol. Next they were digested with 50μg/ml Proteinase K (3oehringer Diagnostics, Indianapolis, Ind.) at 37°C. for 2 to 5 minutes. This step was followed by acetylation andre-hydration of the tissues.

[0162] An antisense probe was generated from the zalpha33 sequence usingSp6 RNA polymerase. The probe was labeled with digoxigenin (Boehringer)using a commercially available in vitro transcription system (Promega,Madison, Wis.) as directed by the manufacturer.

[0163] In situ hybridization was performed with the digoxigenin-labeledzalpha33 probe. The probe was added to the slides at a concentration of1 to 5 pmol/ml for 12 to 16 hours at 57.5° C. Slides were subsequentlywashed in 2× SSC and 0.1× SSC at 55° C. The signals were amplified usinga tyramide signal amplification kit (TSA™ Signal Amplification; NEN LifeScience Products, Inc., Boston, Mass.) (see, U.S. Pat. Nos. 5,731,158;5,583,001; and 5,196,306) and visualized with a commercially availablesubstrate kit (Vector° Red; Vector Laboratories, Burlingame, Calif.) asdirected by the manufacturer. The slides were then counter-stained withhematoxylin (Vector Laboratories).

[0164] Signals were seen in the heart, pancreas, testis, liver, andappendix. The positive-staining cells appeared to be endothelial cellsof vessels, immune cells, acinar cells of pancreas, and spermatocytes oftestis.

[0165] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 10 1 954 DNA Homo sapiens CDS (136)...(672) 1 gccgctgttt tgaaatcgggccgcgggggg tctctcaagc tggttccaac gctgaggccc 60 cacagcctcc caattccgggcagacccctg acacctgctg tctggcccct tccggcctga 120 agctgcagcc gcgcc atg tccacc cct ccg ttg gcc gcg tcg ggg atg gcg 171 Met Ser Thr Pro Pro Leu AlaAla Ser Gly Met Ala 1 5 10 ccc ggg ccc ttc gcc ggg ccc cag gct cag caggcc gcc cgg gaa gtc 219 Pro Gly Pro Phe Ala Gly Pro Gln Ala Gln Gln AlaAla Arg Glu Val 15 20 25 aac acg gcg tcg ctg tgc cgc atc ggg cag gag acagtg cag gac atc 267 Asn Thr Ala Ser Leu Cys Arg Ile Gly Gln Glu Thr ValGln Asp Ile 30 35 40 gtg tac cgc acc atg gag atc ttc cag ctc ctg agg aacatg cag ctg 315 Val Tyr Arg Thr Met Glu Ile Phe Gln Leu Leu Arg Asn MetGln Leu 45 50 55 60 cca aat ggt gtc act tac cac act gga aca tat caa gaccgg tta aca 363 Pro Asn Gly Val Thr Tyr His Thr Gly Thr Tyr Gln Asp ArgLeu Thr 65 70 75 aag cta cag gat aat ctt cgc caa ctt tca gtt ctc ttc aggaag ctg 411 Lys Leu Gln Asp Asn Leu Arg Gln Leu Ser Val Leu Phe Arg LysLeu 80 85 90 aga ttg gta tat gac aaa tgc aat gaa aac tgt ggt ggg atg gatccc 459 Arg Leu Val Tyr Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro95 100 105 att cca gtc gag caa ctt att cca tat gtg gaa gaa gat ggc tcaaag 507 Ile Pro Val Glu Gln Leu Ile Pro Tyr Val Glu Glu Asp Gly Ser Lys110 115 120 aat gat gat cgg gct ggc cca cct cgt ttt gct agt gaa gag aggcga 555 Asn Asp Asp Arg Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg125 130 135 140 gaa att gct gaa gta aat aaa aaa ctc aaa cag aag aat caacag ctg 603 Glu Ile Ala Glu Val Asn Lys Lys Leu Lys Gln Lys Asn Gln GlnLeu 145 150 155 aaa caa att atg gat caa tta cga aat ctc atc tgg gat ataaat gcc 651 Lys Gln Ile Met Asp Gln Leu Arg Asn Leu Ile Trp Asp Ile AsnAla 160 165 170 atg ttg gca atg agg aac taa gctgatattt aaatttcctgctttacacat 702 Met Leu Ala Met Arg Asn * 175 gttataccat tgttttttccctcaagtatt ttttccctgt gaagaagatt atttatctgc 762 ttttatttta gtcactaaaactaaagtttt tatttttaca ttgtgatttt tacattaaaa 822 tattaacttt tttaatgctattttatgaaa gattattgta ataaactttg atggggtttg 882 tattttggtt aatcttcatgaattgaataa ttgttttttt aaagcaaaat aaagtttttt 942 aaataaatgt ta 954 2 178PRT Homo sapiens 2 Met Ser Thr Pro Pro Leu Ala Ala Ser Gly Met Ala ProGly Pro Phe 1 5 10 15 Ala Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu ValAsn Thr Ala Ser 20 25 30 Leu Cys Arg Ile Gly Gln Glu Thr Val Gln Asp IleVal Tyr Arg Thr 35 40 45 Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln LeuPro Asn Gly Val 50 55 60 Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu ThrLys Leu Gln Asp 65 70 75 80 Asn Leu Arg Gln Leu Ser Val Leu Phe Arg LysLeu Arg Leu Val Tyr 85 90 95 Asp Lys Cys Asn Glu Asn Cys Gly Gly Met AspPro Ile Pro Val Glu 100 105 110 Gln Leu Ile Pro Tyr Val Glu Glu Asp GlySer Lys Asn Asp Asp Arg 115 120 125 Ala Gly Pro Pro Arg Phe Ala Ser GluGlu Arg Arg Glu Ile Ala Glu 130 135 140 Val Asn Lys Lys Leu Lys Gln LysAsn Gln Gln Leu Lys Gln Ile Met 145 150 155 160 Asp Gln Leu Arg Asn LeuIle Trp Asp Ile Asn Ala Met Leu Ala Met 165 170 175 Arg Asn 3 950 DNAMus musculus CDS (120)...(656) 3 cactgttggc ctactggagt cttccggtacagcgcttgca tcgcggcggg cggaagtggc 60 gccgcttttt tgaaatcggc cgagtgggctcgcgccggac ccgagccgcc gggggtgcc 119 atg tcc acc cct ccg ctg gcg ccc acgggc atg gcg tcc ggg ccc ttc 167 Met Ser Thr Pro Pro Leu Ala Pro Thr GlyMet Ala Ser Gly Pro Phe 1 5 10 15 ggc ggc ccg cag gct cag cag gcc gcgcgc gag gtc aac acg gcc acg 215 Gly Gly Pro Gln Ala Gln Gln Ala Ala ArgGlu Val Asn Thr Ala Thr 20 25 30 ctg tgc cgc atc ggg cag gag acc gtg caggac atc gtg tac cgc acc 263 Leu Cys Arg Ile Gly Gln Glu Thr Val Gln AspIle Val Tyr Arg Thr 35 40 45 atg gag atc ttc cag ctg ctc agg aac atg cagctg cca aat ggt gtc 311 Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln LeuPro Asn Gly Val 50 55 60 act tac cat act gga act tac caa gac cgg cta acaaag ctg cag gac 359 Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr LysLeu Gln Asp 65 70 75 80 cac ctt cgg caa ctt tct att ctc ttc agg aag ctgcga ctg gtc tat 407 His Leu Arg Gln Leu Ser Ile Leu Phe Arg Lys Leu ArgLeu Val Tyr 85 90 95 gac aaa tgt aat gag aac tgt ggt ggg atg gac ccc attcct gtt gag 455 Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro Ile ProVal Glu 100 105 110 caa ctg att cca tat gtg gat gaa gat ggc tca aag aacgac gac cgg 503 Gln Leu Ile Pro Tyr Val Asp Glu Asp Gly Ser Lys Asn AspAsp Arg 115 120 125 gct ggt cca cct cgt ttt gct agc gaa gag aga cga gaaatt gta gaa 551 Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg Glu IleVal Glu 130 135 140 gta aat aag aaa ctc aaa cag aag aat caa cag ctg aagcag att atg 599 Val Asn Lys Lys Leu Lys Gln Lys Asn Gln Gln Leu Lys GlnIle Met 145 150 155 160 gat caa tta cgg aat ctc atc tgg gac ata aat gccatg ctg gca atg 647 Asp Gln Leu Arg Asn Leu Ile Trp Asp Ile Asn Ala MetLeu Ala Met 165 170 175 agg aac taa agcgatattt aaatctcctg ctctactcatgtgatgaagt 696 Arg Asn * tgggtttttc ccccctcttg agtattcctc cctttgaaaaacgttattta tgtctttatt 756 ttaacagcta gcactaaagt ttctgttttc actttaaagtatttactagc ttttttttta 816 atactgtggg ttttatgaaa gattattgta atacctttgatagggtataa attttggtta 876 atcttcagaa attgaataaa ttaaaaaata caaataaaaatttaaaaaaa aaaaaaaaaa 936 aaaggccaca tgtg 950 4 178 PRT Mus musculus 4Met Ser Thr Pro Pro Leu Ala Pro Thr Gly Met Ala Ser Gly Pro Phe 1 5 1015 Gly Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu Val Asn Thr Ala Thr 20 2530 Leu Cys Arg Ile Gly Gln Glu Thr Val Gln Asp Ile Val Tyr Arg Thr 35 4045 Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln Leu Pro Asn Gly Val 50 5560 Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr Lys Leu Gln Asp 65 7075 80 His Leu Arg Gln Leu Ser Ile Leu Phe Arg Lys Leu Arg Leu Val Tyr 8590 95 Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro Ile Pro Val Glu100 105 110 Gln Leu Ile Pro Tyr Val Asp Glu Asp Gly Ser Lys Asn Asp AspArg 115 120 125 Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg Glu IleVal Glu 130 135 140 Val Asn Lys Lys Leu Lys Gln Lys Asn Gln Gln Leu LysGln Ile Met 145 150 155 160 Asp Gln Leu Arg Asn Leu Ile Trp Asp Ile AsnAla Met Leu Ala Met 165 170 175 Arg Asn 5 178 PRT Artificial Sequencevariant polypeptides 5 Met Ser Thr Pro Pro Leu Ala Ala Ser Gly Met AlaPro Gly Pro Phe 1 5 10 15 Ala Gly Pro Gln Ala Gln Gln Ala Ala Arg GluVal Asn Thr Ala Ser 20 25 30 Leu Cys Arg Ile Gly Gln Glu Thr Xaa Gln AspXaa Xaa Tyr Arg Xaa 35 40 45 Met Glu Xaa Xaa Gln Leu Xaa Arg Asn Met GlnLeu Pro Asn Gly Val 50 55 60 Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg LeuThr Lys Xaa Gln Asp 65 70 75 80 Xaa Xaa Arg Gln Xaa Ser Val Xaa Xaa ArgLys Xaa Arg Leu Val Tyr 85 90 95 Asp Lys Cys Asn Glu Asn Cys Gly Gly MetAsp Pro Ile Pro Xaa Glu 100 105 110 Gln Xaa Xaa Pro Tyr Xaa Glu Glu XaaXaa Ser Lys Xaa Asp Asp Arg 115 120 125 Ala Gly Pro Pro Arg Phe Ala SerGlu Glu Arg Arg Glu Ile Ala Glu 130 135 140 Val Asn Lys Lys Xaa Lys GlnXaa Xaa Gln Gln Xaa Lys Gln Xaa Xaa 145 150 155 160 Asp Gln Xaa Arg AsnLeu Ile Trp Asp Ile Asn Ala Met Leu Ala Met 165 170 175 Arg Asn 6 534DNA Artificial Sequence degenerate sequence 6 atgwsnacnc cnccnytngcngcnwsnggn atggcnccng gnccnttygc nggnccncar 60 gcncarcarg cngcnmgngargtnaayacn gcnwsnytnt gymgnathgg ncargaracn 120 gtncargaya thgtntaymgnacnatggar athttycary tnytnmgnaa yatgcarytn 180 ccnaayggng tnacntaycayacnggnacn taycargaym gnytnacnaa rytncargay 240 aayytnmgnc arytnwsngtnytnttymgn aarytnmgny tngtntayga yaartgyaay 300 garaaytgyg gnggnatggayccnathccn gtngarcary tnathccnta ygtngargar 360 gayggnwsna araaygaygaymgngcnggn ccnccnmgnt tygcnwsnga rgarmgnmgn 420 garathgcng argtnaayaaraarytnaar caraaraayc arcarytnaa rcarathatg 480 gaycarytnm gnaayytnathtgggayath aaygcnatgy tngcnatgmg naay 534 7 6 PRT Artificial Sequencepeptide tag 7 Glu Tyr Met Pro Met Glu 1 5 8 39 DNA Artificial Sequenceoligonucleotide primer ZC23310 8 cacacaggcc ggccaccatg tccacccctccgctggcgc 39 9 39 DNA Artificial Sequence oligonucleotide primer ZC233119 cacacaggcg cgcctttagt tcctcattgc cagcatggc 39 10 20598 DNA Homosapiens 10 ctcaacaatc attcaataag tatttattga tacttgtgcc aatcactaggtgttgagaat 60 aagtgaataa aatagtctgt tttcatggag ttggcagtgt gtgtagatagggtgagataa 120 agtctgaaag taaacaaact gaaagctata taaaatatca gacagtaggccgggcgcggt 180 ggcttatgcc tgtaatccca gcactttggg aggccgaggc gggcggatcacgaggtcagg 240 agatcgagac catcccggct aacacggtga aaccccgtct ctactaaaaatacaaaaaat 300 ttgtcgggcg tggtggcggg cgcctgtagt cccagctact cgggaggccgaggctggaga 360 atggtgtgaa cccgggaggc ggagcttgca gtgagccgag gtcgcgccactgcactccag 420 cctgggtgac agagcgagac tccatctcaa aaagaaaaag aaaaaaaaatcagacactaa 480 taagagccag agtcagaaag aagtgtgtgt gttgatcatt ttttttgcttacagatactg 540 ggaaaactca aagaataaaa catctgagca gggaccaaga gcaatcggggagtccgtatc 600 atgtcacaga gaagcgtaca agcttaggaa tcttgttttc tgaattcgaatcgcaggttg 660 ccagttacac ctgtatgcga ctcaacaagt actttaacct gtctcttagctgttatacta 720 caaacctcag gcttgttaag agaaaatgct ccgcaaggaa ctaaacacagaaggttctaa 780 cgctgagcca agactgggaa acgaactctg ggaactcacc ccaggctccccaagaacatc 840 gcccctctgg ctggagcgca attggtgatt ggctacttaa cccgtccgtcctttcccgcc 900 caggggtcca atccaatcca gcccggctcc gctcggagac agttcgccgagtgggcggtg 960 tctatgacgt tttctgacgt gttacgtcac agtgggcgga agtcgcggccgctgttttga 1020 aatcgggccg cggggggtct ctcaagctgg ttccaacgct gaggccccacagcctcccaa 1080 ttccgggcag acccctgaca cctgctgtct ggccccttcc ggcctgaagctgcagccgcg 1140 ccatgtccac ccctccgttg gccgcgtcgg ggatggcgcc cgggcccttcgccgggcccc 1200 aggctcagca ggccgcccgg gaagtcaaca cggcgtcgct gtgccgcatcgggcaggaga 1260 cagtgcagga catcgtgtac cgcaccatgg agatcttcca gctcctgaggaacatgcagg 1320 taggaaggcg ggcgcgcgag gccaggggga tgcagctggg agggaaagggcctctggttt 1380 cctctttacg tggggcccgt ggatgtcatc ggggctgccc tgggcaaaccttaggtgtag 1440 ggtctccccc attcacactc aagagtgtcc ccaaaagtcg gatttgtagaggtgatgttt 1500 tccagaccag atttctgcag aattgaagcc aatttttttt agattcaatcgagctcttta 1560 gttactctgt aaactcttgc acctttcaag gcagagatgg gtcgcactccacttccctct 1620 ttaggttgct ttaaatgttt tttccagcat ttcttctggg gaggtccaaggagttgaaga 1680 atttacattt tttttaaaaa aacagtcctc ctttttttgg catagttaatatacagtaaa 1740 ctacactcac ctaaagtttt gatatatatg tgtactgtag tctgtatacttgtgaaatta 1800 tcaccacaat caaaatagtg tacacatcat cccagaaagt ttcttcatacccctctgaca 1860 tctttgcttc ccaccttcca tcgctaagca accattgatc tgcgttctgtaatcataaat 1920 taatttgcat ttgctagggt tttatataaa cggagtctac agtatgtgggcttttttttt 1980 tgtctgagat ccttcactaa gaatgcatca ataatttcct ttttattgctgagtaatgtt 2040 ccattgtatc catgtatcac catttgccta ccttttcttc tgttgatggacatttgggtt 2100 gtttccactt tttggctgtt gtaaataatg ctgctatgaa cttttgctgacaaatttttg 2160 tgtgaacgta tgtgtcattt ctcttggaca gattcaggag tggaattgctggatcatgca 2220 gtacatgtct gtttaatact ttaagaaact tttccaaagt ggcctgagccattttacgtc 2280 ccgattattg ctagtgggaa tgtaaacatt gctgtttgag ggttctagtttctcctcatc 2340 ctcatcaaca cctgttatgt cttgatgctt tccaaatact tctcccagtctatcctgcct 2400 ttttattctc ttaatactgt cttttgaaga acagacattt taaattttgatggaatccag 2460 cttactgatt tgttctttta tagattttgg ttttgctgtc atacctaagaaatctttgcc 2520 taacccaagg tgacaaaggg tttctgtttt cttctagaag ttttgtactttcaagcttta 2580 tatttaggtc tacaacctat atttagtttt gtgagttttt aaatatgtatacatagtggc 2640 agatacggat ccaagttcat tgttttgcat ttggatatcc agttgttccagcaccatttt 2700 gctgacccat ttttaatgct tgcttatatt acccactaca tgtagtagatattatttttg 2760 ttttacagat gaagattctg tacttcagag agataaagtg acttgcccaaacctagtcag 2820 tggtagggta aatttgaaac caagttttca aaaatcttac agctcttatttctgtgggtg 2880 tccaaaaaag tgttttattg cttttttttt ttttaactgg tcaagactttttttttcgtc 2940 ctgattataa gagtaattca gtttcgtgga aatttttaag accatgtgaaatatagtaca 3000 aaggagatcc aggagaaatt atgacattct ttggcaataa tttaaaaaaatgaatagatt 3060 catatctaga atgaataaag tacggattaa ataatgtctc agaatttagcatactttaaa 3120 aaaaattttg tcctgttata tgcaaagcac cgaataatac ctaacacttacgaagtactt 3180 tccttggtca ggtaccctgt tataggagca gttttaagga gtatttttatccctatttta 3240 cagtaaagga aactgaggct taatgagaga aaaaataaat tacccaacatcacacacgac 3300 taatagctct tcctactaac ctctttttaa tactaacttg ctgtcaatgtacattattaa 3360 aagatataat acaggttcat gttctacgca gaactaaact ttttcaaaaaaagtatttta 3420 tcaataacta gattacatgt ggacctgcat tcaaagccaa cttttttttttttaagtttt 3480 ttttttttgt ctgtaagttt attcaatgca aaataatcct ctccaattttactgaggtgg 3540 ctgaccatgt ccacgaccaa atccgcctct aaactggaat tcggttgttgacccagcccc 3600 agtctcggct ttcttgtcgg caccaggggg cacagcactc cgtctgtaggtatctctgtc 3660 ggcttcccct cttgtgagtc ttgcaggtcg ctcaccctcc agacctttaggctgaggcct 3720 gccagtctct ggacggctgc ggcgtagggt ggcaggaaca atctccgggggcagatgaag 3780 gtaatcacag agatactgga taccctcatt ggtaaggtac cagtagaaatgtctccaggc 3840 aaactgttcc ttcacgtagc ctcaggactt gagaaactgc atggccttcatgacatgaag 3900 gttggacaca ttcttgtctg ccagctccgg gtgcttaggc atgtggaatcctccttggcc 3960 accatgattc cctccttaaa aaggagttca taggctaggc gcggtggctcacgcctgtaa 4020 tcccagcact ttgagaggcc gaggtgggcg gatcacgagg tcaggaattcgagaccagcc 4080 tagccaacat ggtgaaaccc catctctact aaaaatacaa aaattagccgggcgtggtgg 4140 cgggcgcctg taatcccagc tactcgggag gctggggcag gagaatcgcttgaacccggg 4200 aggcggaggt ggcagtgagc cgagagatcg cgccactgca ctccaggctgggcgacagag 4260 cgagactctg tctcaagaaa aaggagttca taaatggcaa tacgtttcttcttaagcttc 4320 aacatttcgg ccactgtagg tctgggaccc aaagccagct tttgagggactatgataatt 4380 atatgtgtgc ttttcatttt gtgaaatctg tgatcactca tccaggacctttggggccta 4440 tcataattgg ctgaaaagat aacaatgggc tccttcattt ccaaaaaaactagaagaggt 4500 gatagaaata ggatattctt aactatatat aacctagtga tttgttaatacatttttagt 4560 attcacattt taaagtaatc gtctgtatta ttgatctacg agagtaaagtgctgtgaata 4620 caaactgcta agaatttttc taagcatttg ttttaaagaa gtggttcctggtatacacta 4680 gatgtccgtt agccacttga gagtctgatg aaaactgtga gctctcttctgagagaaata 4740 cacatacatt ttgaaaattt caggggttaa aggaagattt aggatactccccaagtatcc 4800 ttaaatctca gattaagagt ccttgtggaa gataccattg cttttgttctgtttattatt 4860 ctttgaatct ttaattttta aaatacatag tacattcaga atttaggaataaaaaaggac 4920 agtgcagtga acattttcct tcccattcct atctcttata ctatgtacctaactcctctc 4980 cacaggcagt gattgtgact gattcttact atttttgtgt atctttccagatattctctg 5040 catattaagc aaatgtttat gtatattgtt tcattttcgc cttttttgcacagataatag 5100 tatattatac acaaccattt tgtaccttgc ttttttcagt tgatatattttggaatttat 5160 tcatacccat acagaaaata ctcccttatc ctttttatag tgtatagtttacactgcatg 5220 gaaatactgt aatttattaa accagttctc tagtgagggc atttggctgtttttcatctt 5280 tccctgttag gctgcaccaa ataacctcct acacaaattt gcacgtatacctgtcggata 5340 cgtttttaga aatagctatg tgcttttgta attagataca ttttgatgaattgtcctcca 5400 cagagattga actaatttat gctacggcct aagaccagca cagtgagttatagctttttt 5460 ggatctttga tcacttgata ggtgaaaatt ctactttaca tttctcctagtttgactgag 5520 tttgaacatc tcttaaaata ttttaattat ttatattttt ttcctgctaaatgcttgttc 5580 acttattatt cacccatttg tctgtctttt tttttttttc atcttttttacctaccttaa 5640 ttgtaggatt tagctattta catactagag aaacctgctc tgtgtgatacaacttaaagt 5700 tatttttttc cctgtctttt accttttggc tctgctaatg gtagttttgccatgcatttt 5760 ttttaagtag ttgaatgtat tagtctttta ggacttctag gccttatgcctagttagaaa 5820 ggtcttcccc actcaaatta ggaagaattc tataattttt aaaatttcagcctttacatt 5880 taaaactgtg atccttatgg aatttatcct ataaaatgtg aggtctgggcctagccttaa 5940 ctttttcaaa aggttcactg aataattcac ttgcttcttc cagtgatttcaggtagcttt 6000 tatataagtg aatttgtttc tggatgttct attctattcc acgtagctgcctatctttta 6060 tgtattaatg caaaacattc ttttaatttg tcaggatcta aatgtgttacacttattttt 6120 cagaatttcc ctggctattc ttgcttattt ttccacatgt attttagaatcagcttattc 6180 taagaaaaag cctgttagca tttttattag gtccgttcgg cctagaaagacttgacatca 6240 ttatgatgtt gactcttact aaccaagaac atgcagtatt ctttttctgttttcctataa 6300 ggtatcttaa gttttcattt ttatcttgag attttaaaag tttttttctaggtattttat 6360 cttttctgtt gccattgtaa atggggtctt cttttcccat gtttttgtttgtgttataga 6420 tgaaaacaga gctattgctt tctctttgga cctacttcct tattgaattcttttattttt 6480 agtagcactt tatcatatta tttgtaaata gattatgatg ctttctttttaatgtttcaa 6540 catttttttc tctttcttct ctaattgcat tggctagtgc ttccagaacagtgttcactt 6600 acagaattag ggtcttagta gacatccttt tctgtttctg atcttagttggaatgcttct 6660 aatgattcca cattaatcat agtgctgact ctttaactga aataaatgtatgttgtcaca 6720 tattgaggat tttttattga gttttatatt gattttttaa atgggtgttgaatttagttg 6780 aatgccttct tggtgtctat ggatacgatc atgtatttat gggttttcatccatactcat 6840 gagtgagatg gttattgatg agcttttcag ctgaacagct acaccgtggtatataccaag 6900 ggatcttagg catttttacc gtgtatactc tgtactatgg aaaccttgtctctctgatgt 6960 ttactgctct atcctagctt ctagaacagt gctgacacat gaaacgtgtgtggtattttt 7020 tctttttaat gatttagtaa gttaagttag taagtttgga tcttagatttaacatctaat 7080 atctttctaa gtcagcaaat ttggggagtg tgattcttgg catttcaaaaggatttttgt 7140 tttctttatg acagtattta tcaagagttc ttttacataa tacatcttaataaattttaa 7200 gtatagattt atgggtaaaa taactcgctt aagtaaaata attcacactgtactttaaag 7260 ccttctgtat gtgtgcacta tgctcaatat ggtgatatga acctaagttccagtctttac 7320 taagtagaaa atatgggcaa agtattaaac cccataatgc cataataaaggtacagagtg 7380 ctgttctagg caagacataa tcatcatgct cctttctgtc acctaaacaagaaagcaagg 7440 atccacttaa acttttctct gaatttctag ttagtgtctg tggaatttcataaactgaga 7500 gttcaggagg agaaatctta cattgtactg agaaatgaac ccttaatgcctttctccttt 7560 aaaatgtttt gactagttca gcctttgata ttttatcaca ttgctttgaggcattgtaga 7620 ctgttttaat gcttgagaat ttctgaaact cctaataata tcacacttcaaataatttca 7680 ggagatccat ctccttaact tgtaaaccat aacagtactg gaaatttagaagtatgagaa 7740 aagaagaaaa taaattattc aagcctattt atcttacaca tcttttatcaactatgtata 7800 gggctttcca gtcttttttc atgtttatgg ttttaatcat tatatgtcctgtttactttt 7860 cactccaaac attgtatcat gcttttcttt ttttctttct tttttttttttttttttggc 7920 attcctacct catgtgttta aaaaagaaaa aaatgcaaac tgaggcttggaaagactgga 7980 ctgatcacct agctattata catgtgaatt cctacaaatg aaaatgcccgaatactagaa 8040 aacgtggtac tgtgtaaata gtattgattc aagtattagg gtgatgacacagatcagatt 8100 gtgggaataa aatattgtac agctgtacag agaaagattt tgttcatgagtttcagcaat 8160 gcaaagtaca tatttagtta taaattctaa ataaatagtc gatttttgtttacctgtcat 8220 tttattattt caatatatac ttttcttctt ttgataaaat tcctgaattaaaaatatatc 8280 catatctaag gcagagttat gatcatagca gttacttgta ataatttattaatggtgttc 8340 aagtatagat ggcacccata tctcctgcta ttgatttttt taagtaaaactttaaatgac 8400 aggtttatat tttaatgtat gccttaagca tcttaattat cttatcagagttaaggcatt 8460 ttgagaatat ttttgcaata ccagccatat attcttcatt ttcttccactaaaatatcat 8520 cagattcact ttttttttta aactgtactt tgtttttaac ttcttggttgaggtcagcag 8580 catgctgcat ttatataata gtaatattaa aattgtaaat ttttttttaaagttacactt 8640 caaaaattaa aggggaaaaa gtatttcaag tcagagggtt tatcacaaagtttttctagg 8700 tctgttttat agcagtgaaa caggtatgcc aagttgttaa taaatgggcatgcattgctc 8760 ttaaattctt aaaattaaac ttgatcagtt gtgagggaca gcttatgtctatgcattgcc 8820 taaaaattgt ttctgtttgt tggctttact tatagaaaat cttgatttttttttcattac 8880 ttgatatttt tattctttgt ttttcctgat aagctgccaa atggtgtcacttaccacact 8940 ggaacatatc aagaccggtt aacaaagcta caggataatc ttcgccaactttcagttctc 9000 ttcaggaagc tgagattggt atatgacaaa tgcaatgaaa actgtggtgggatggatccc 9060 attccagtcg aggtaatttt ttgtgataga gggaggatga atataaggtgtgaactagtg 9120 tcaagacagt tgttaatctt tttcctctct ctcctgtttt agcaatatttccaggtttcg 9180 aattttattg gttttacttt tgaccagaga agatgaaaat gtgatatatatgcataaaat 9240 actactatat ttttacatta acaatttctt gtagactttg acaggcttatagtaattttt 9300 gttgttgtta ggaagaaatg ttttcctatg gcaattttta ttgctgtcagtgtctccttt 9360 taattaagtc agggatattt ttagtaggaa ttttaatagt atcatatacttttaaggtgg 9420 ttttcatgaa gtactttaca gataaaatat tatttttatt attattaatttggattttat 9480 atttagccca gtggaatcac tatattttaa aaatatattc tctaaatttaatcttgggtc 9540 atacctattt agttaattag gcatatattt ctataagtca ccctttcctgttgacaaatt 9600 atatctctgc caccaaagga agctggtaag agatgaccag agaaaaattaaactgcaaaa 9660 atgcctaaaa agaaggggtc agagatgtgg gagaaagatc agaaatgtggcctacatcgt 9720 cagatgctac agagaagtca gggaaaataa ggaatgaaaa ctgtccagtagattttgcta 9780 gatgaaaatt gttggtatat ttgagtagag cttcagtgaa atgatagcagcgtgaagtct 9840 gatcctagtc gggtgaagat taaacaggaa ataagaagct ggagccggcaagtggatatc 9900 agtctttcaa gaagctttga aaaaaataag agaggaaagc agtagatggaaggagagatg 9960 gaatcgaagg tttgcaggag gaagaggagc ttccctccaa atagaagagataggaacatg 10020 tttgaataat aaaggaagaa agctgtaggg agaggttgag agaaaggtgtataaataatg 10080 aagacaggag ggcagagtaa tcagggcaca ggaatggaga ctaatcgtcttcaggaggaa 10140 ggacagctct gcattgagac agatgaaaga aagaaaagga gaatgtagacatgggtgagt 10200 ttatatatat cccaagaatt tgaagaagtt ttatcaggtg aatcctagttttaccagtca 10260 tttaccttta gcctattctt gcattggtaa tacacgatgg taaaggctaggtgattcctg 10320 aagtctgcca cacttatgcg gtaaattcca tcactaacca tgcatacttaattacgattc 10380 tgctagaagt ttcattgcta tataaatttg tttctcctac aaagaaaaagttctcacaac 10440 tgcgaaaact taagacatct agaagtttgc atagactctg aagttttagattgcaagtta 10500 atgacaatta tataatacag atccgtgaag ttcttttctt tttattgcagatgaaggaaa 10560 gcctgtttta tctatctcaa tagactacaa atctcttagg ggcaggaatcacatcttgtt 10620 tggcattact tttatagtgt ttcataatta aatatttgct gattgagtagggttttttta 10680 aatttttatt ttattttatt ttatttttta cttctcccat cttcctgctggaagtattgt 10740 ttagttgtac atgacaacca atgttataaa ggctgttttg caaaagaaacctgaaagggt 10800 aatgcataga atgcattgaa atggtcaact attaaagctc ttatttcaggccatcacctg 10860 aattgaaatt atttttatac aaataaaagt ataaaaattg accttaatctgatatattct 10920 ctatacaagt gattagaaaa cttgaattat atttttgcct tttaatttttgttaatcatt 10980 cccagcaact tattccatat gtggaagaag atggctcaaa gaatgatgatcgggctggcc 11040 cacctcgttt tgctagtgaa gagaggcgag aaattgctga agtaaataaagtgagttgtt 11100 agtttttaca ttttatgttt tagagttatt gataaaaatt actagggagtctgatgtaac 11160 tccaaaacat aatttgaatt atcaaattac atgaagttgt ttctgcggcaaacttcacct 11220 atttagaata cagccaagaa atgaaaggta agcagaaatg ctgtctgaatagtcagaaga 11280 gaaattgcaa aattcattcg ataaagctct tagaagagct ccttagacacttagtcacaa 11340 tccatttact tttttctttt ctgggcagaa ttctaccttt ctagcttatagcatatttgg 11400 agcagcaaat gataaaaatt tacaaatatg aaatgtgaat gtctcaacacctgaataaaa 11460 aatttagaga actatcccag cattattatc tactccaacc ccacaaaaagcttgtaactg 11520 gaatgtattt atcgctgaat actttcaaaa gttcagaaga taatgttcaaatagttctcg 11580 tgaaacagag agaaataggg aaaggtgtca gttcattctg gggatggaaatgtggcccca 11640 gcttagcaaa acctaccaga gatagcacag tgaataaaac tttcagcatcactctttcac 11700 tgacaaatat agattgcccc ttcctgaaat tcttaattct aattaatagaactcatcagt 11760 gtattaataa tccaccactg ccaaagagag tcttctgagg tatatgaagattttatagtt 11820 cagtttgtat gtataataca tgatgtcagt aaactacctt gaaaaaagattttctcacat 11880 gtctaaaaga catctgataa aattcaatac tgttgctgat tcaaaggacacttaaaacta 11940 ttagcaagac agtgtgtttt ttttttaaat ctttttcttt tagataaatacccaacattt 12000 ttcataaggt gaaatataga aatatcctta ttaaaaacaa gcatgaaataagatacttta 12060 taacactatg tttaatatta ttttagaagt tatagccagt acaaaaagacagtggacaaa 12120 tgacagttgg gaaacaggag ttactatgta tgagcttgtt agaatggtcaaagaaactca 12180 actgatgaac aactagtcta taagagttcc gtaaaagggt ctaagacaagactgatacca 12240 tgctcttaca atgctgtaaa cctattagat tataagattc tattgactcttgtaacaatt 12300 aatattaagt agataggaat tatgtcaata atgtgtatga ataaacttattaagatacat 12360 aaaggaagct ttgaatgatt aaaagaacat ataaagttct tggatgagcaatacatatta 12420 taaaaatacc agtcttgatt taatactcac agttttaata cagtaacaatccaaatccta 12480 ataagaaagc atgcaaaaac aataggattt tagtgcaata ccaatcaataaacaaatcca 12540 aataaaaccc aaatggaatt tttttcttga aagtatacaa aaatatcaaagaaaatgttg 12600 ataaatacga ctaataaagc agaattctta gcttcaaaaa ttaaactgtaatatgaagca 12660 tattataaaa atggtaatta ggtgtataat actggcacaa gaataggcagattagtaaaa 12720 taatctagcc agaaaaacag ccttcttatt gtacatgaga atttaatatgacagaagagg 12780 gcttgtgtgg aaaattagtc tttagaaata gaaactttgg gggcgaaattaagctagctt 12840 ttacattaca ccataaatca aaatgaattt cagagttgaa aataagacctgcaaaacaaa 12900 atattggata attttttaag tattttaaaa ctcggaagaa aggaaaaagatggatagatt 12960 ttataaaata aaattttgta acttttttaa aaggtgaaac acacatttagaaaactattt 13020 ccaaaaaatc tgattgtaat acatgacaat cataataaaa aactcacagtaaaaaattgg 13080 tagagggtga ggaggagaat gggaactttt agtttagatc ttgaaaaataactggtgttt 13140 ggtagtctag atgtatgaga cattcactta ttaagacgtg ttatatgtttctgttgataa 13200 atatgtatta cctcaataaa tgtgttctgc ttatgtttct ctaatgatttttttataaaa 13260 tataatgaaa tatgtatata aataaaacac cattataata ataactttggtttcagagta 13320 ccacatgtgt aactaatgta aacatagttt gttctgtacc atagaggattttataatatt 13380 ttgtaaaaaa taaagctgtg taaattcaga aagatacttt ttctatatttacatttctct 13440 tcaattttca ttaagtttct cttttttccc aatttgataa ctcagaaataacatgtcttt 13500 gacttttaaa agttctcttt tataatcaaa atatggtaat tctttggcagcatgtaacca 13560 aaaaaaaaaa tcttactatc tactgacata gagttttgtg gattttaagtaaaggatata 13620 attgaaacaa ggatatgatg tataaaagga cttctttctc cccatgaggatttagctccc 13680 tacagcctct ctctcctgga cccacctaca taattgtttc cctcaaacctttcagtgcac 13740 ttatatcata tttggggttc agtaaataat ctgtatcata taattatggcttcataagta 13800 ttgtttattg ctaagccgca tagtatacta gggtcatatt tccttgattgaataattttt 13860 tgtttttctg aagttaattc ttacctcttt aattttctaa aattttagttagttttttat 13920 gcatctatga caacatctca gtactatttt ccaattttcc acagtcagaatcatcagaca 13980 gtttcatggt tctcatccca ccctcccaag ttttctatgg tcttataggtctggatggaa 14040 tgctggctag gcctgctttt cagctctggg gctttccttc acctcttctgttggtttcac 14100 tatttcttca gctcatgtct tcccttttct tggttttctt ccttgttggtgatgtgcata 14160 catcagtgac ttctttagaa ggaatgcaga cacttaaatt ttgagtccttacatatttgt 14220 ccagttgata gtttggctgg ctataaaatt ctatgttgta agttatgtttcctcagaaat 14280 ttgaaggcat tccataatct agtatagggt ttggcagact ttttgtttaaactactagat 14340 agtcaatctt ttaaccattc ctccagtttt cattcccagg gctcacctggtgcctctgag 14400 atggtctctg tctgtggttc tccatggtgg atcaggcgta ttctcggtggcatccctctt 14460 gcatgcccag gccttctctg agtcactgac cactccctct ttacttttcatctgaaaatt 14520 taccttgcat ctgctgttgt gtcctttctg atcacttttc tctgcatcagtttatagctt 14580 tttttattcc tctcctgcca cacactcaat agatacacaa tgtttctgaaagtttgtgta 14640 tatttacgga atgaaagtat tttaaatttt tttccccatt tttacttctgacattgagat 14700 ctcttccttt gattaaatga cttggatctg ttctaagtgc ttttaacttcaggataaaac 14760 atgttttagt taacgtgata tcaaactgat gggttattac aaagagaaaagaatcagttt 14820 aggtgtttta aatgaaccag gtttaaagct aaactctatt tctgtgggaattgcttttta 14880 aaagacaatt tagaggtaaa gtaccttctt gagtatcatg gggaagaactcatttgatga 14940 cctcataact ctctttgtat gaattcttct acctatattt tgagaaagcactgaaagatt 15000 aggtggtggg cacagggaaa agatgggaga gggctaacat ttggttaacttaaatttaaa 15060 tcccaaataa ggaagaagaa aataaaatta actgctaaaa ggcaaacaaattgttttttt 15120 tttttttttt taccaaaagg gctttttaat tcattcctta aaaaaatatataaatgtgaa 15180 ttaaatgtag catattctgt tactagaata atcttaccta cagttaaccctagctactga 15240 catagagttt tgtggatttt aagtaaagga tataattgat acaaggatatgatgtataaa 15300 aggactttct ccatatgagg atttagctct ctacagcctc tctctcctggacccacctac 15360 ataattgttt ccctcaaaca attttatttt attattttcc tcaacttctttaagctctgt 15420 gtctccattc tagaatataa gattaaaata tatgattaaa agttttaatttttaaaatac 15480 tgagctcgtg atccattgat actttttttt tttttttttt tttgagagacaaaatctctc 15540 tctgtcaccc aggctggagt gcagtggcac gatctcggct cactgggttcaagcaattct 15600 cctgcctcag tctcctgagt agctggggtt acaggcgtgc accaccatgcccggctaatt 15660 tttttgtgtt tttagtagag atggggtttc accatgttgg tcaggctggtctcaaactcc 15720 tgacgtcgtg atcacctgcc tcggcctccc aaagtgctgg gattacgggcgtgagctact 15780 gcacccagcc gatacatctt tttttttttc cgtaatggca cacacacccgagggtgtttc 15840 tcgtagtgaa aactggctat agatgataat gtactgattt gattatttaatcacatagat 15900 gctattaaaa ataaagttca ctgatttgat ttgaccacta tttataaatagctacattca 15960 taaacttaag tttttgtctc tgtcaattat gttaatccac attcttatgtaaatataggt 16020 ttgctttttt tttagttcat gcatttacta ggtgatagag ttttaaagttcacggaatct 16080 tagctggtaa cactaaaaca ttgaaaatac atcttaccta ttatggtatggagtataaaa 16140 tgcatagttt ttaataatgg ccattattaa atgtcttttt tttttatactgaggagttat 16200 ttaaacaaaa ataagttttg aaatgattgg caataatttt tgtttgcttgcttatttttt 16260 ttccattctt gtaggttttg ggagaagctt tttgatttgg cttttgttattttttcttca 16320 aacatgaaaa tatttaaata tttgtgtgcc tgctctctga caggcattgtcctcagtacc 16380 agaatttctt ccatgaagca gctggatgtg ttgtcggcta tatactcatttcacatgagt 16440 ggacaagttt cttcttggag aacataagac tccagtggaa acaaagcagcacctgtttca 16500 tgtacccatt tcatgttttg atgaatattt acagaaacaa aagtattgtgccatattttt 16560 aatacattgg catttggttg tattaaataa gttgctttat agatctgtgtaatttataat 16620 aaaattatat tatttgcatt caattaaaaa gtcagaattt tattacaatgaatattttct 16680 ttctttctaa ataagctacc taaaataagt agatttatat acccctcagctgaccctcag 16740 tcattttcaa gatattatca tatcattgtg tacttgaaat tgtgcagaagaacatattga 16800 tggtgtgttt actatttagt cgttatggtc tgtctggttt ctaacaatacaaaaaccacc 16860 aataactatc ctggtgctat gaaactttga ggcacatgat ttgtagtcagtatttgatgt 16920 aaatcatcat aagacaatat ggctggtttt aatagtcaaa ttcgtggtttatttaaatac 16980 ttttcatttt ctcttaatat atcctggaat ccttaagcca aatacccatttaaaggatgg 17040 acacttgttt gagaccatat gctttaggtg caggggtgtc caatcttttggcttccctgg 17100 gccacattgt aagaagaaga attgttttgg gccacatata aactatactaacattaatga 17160 tagctgagaa gccaaaatat aaaaaaatca cgaaaaacat ctcataatgttttaagaaag 17220 cttacaaatt tgtattgggc tgcactcaca gccgactggg gccacattgcctgtgggcca 17280 cagttggaca agcttgcttt ggggacatca gaatgagttt tattttgcttaacgctcttt 17340 tcttgatggt aaaatgaaat tgaaaaagat agtaggtagt atttacaaggttggcattga 17400 gtccatgcat ttagaaagta aaccattttt tgtcaagtgt cacagcttttgtgtgaatat 17460 tacattacat ggtatcagtt agcatcaact ttttttaagt atttagtctacttacacatt 17520 cgcctgttaa tgtacaaaat tgggcacagc tgatggattt tgaaaatagcagctgtcaac 17580 gtctgttaaa atcaatcagt ggcaagactg tgtatattaa atcctatatcgtgtcatgaa 17640 aatttattta cactttagcc tctaataagc ataagtgtca gctttgctgttgatcatttt 17700 catgacatgt tttgactctg gatattttat ttgtatgcta atttttttaatttaaaactt 17760 tgtggcatct ttgagaaacg ctatcccaag agccttataa tatattaattatttaaaaat 17820 tttctattcc ccagtgttga atattatctt gaaaataccc tacaaattttagttgttact 17880 tttttaaaaa acaattacga ggcttaaata aaaaagagta ccagaatgtatatttccaaa 17940 tgaggattct tgagagtagt cttattggta ggttatgcat ttatttcagtggtgagacca 18000 ttgtgatagc cagctgcaag ccctgagttg atactgatta taaagtctgtttttaaccac 18060 ccagatgacc aattgtcatc ttatgtatgg gtatgcagaa agtacaactggcagagacct 18120 tagattttat aatccaatta cataaatgag aaaaccaaga agaaggaatttatctaagat 18180 caaataccaa gttagaggca aagttggaac tgggaccgag attgccagcctcccaatata 18240 gcacttttat attcctctgt tttgctgcta ctcttgattg ctggcagaagtgttggtttc 18300 ttggtctctg tgatgaaaaa gactttatag gcttggttat tcttcatctttcccagcaat 18360 ttgaatcaat tttgttagat atatacaacc acttaaataa cagactatcacttacttatg 18420 ataacagaat atagaagatg ggtcatctga aaacttgtgc aagggagactaactaaaaat 18480 accagcagtt tttgtttccc gaagactgtg ttaaagtgag ctttagagtcgagccctgtg 18540 tactgtatag tcctgttttt ctctgactgc attgtgctag ttctcttttcaaatgtgaat 18600 taaattcagt tatgaaagat tattaaagtc tctgtaaatt gattttctattttctaataa 18660 gagtttcttg gtctttcctt ttagccacct atttcttgtt acttccctaactctgaatag 18720 aagcaataca agcaacttca ttttagtttg ttttttaatg aaatcaaaagtaagattgtt 18780 aggcctcttt ttaataagca tacaccaatt tatctggagg tagtttaatatagtgtctta 18840 aaattaatta agtatattat ctccaaatgt aatgtttcca aggggttattgaataataat 18900 aattattgtt attataacta gtaaacactc tgaccagaag ttctctatttctatcttgtt 18960 ttatctaagc agacatttac cacataggat ttgaagaaag aatggttaaaaagaagtcta 19020 caaatcttag tcaagtagaa tccaagtgta gagatacaga gacacatgccgtgcacattt 19080 caggcattca gcaaatctta gtagagaaga ttgcattggg tactctgccatttgtcttca 19140 gtttttagaa ttagaaggag attgcaattc atgctatagg caaacagtgatttctgccgc 19200 acttttgaaa gactgattgc atgaggcatt gtacttttat tatattttctatatcgacag 19260 tttacatttt gtgcctaaca gtaacatttg ttgaatgttt acttatgtgtcaggcacaat 19320 tctaatactt gacatgaatc agaccattta atcttcataa cagctcttatgaggtagttt 19380 caattattat ttccattttt ctgatgtgga aactaaagta ccgggaggttaaataattag 19440 gtcagtgttg taagtttcat agtgaaggat ctggccgggt gtgatggctcatgcctgtaa 19500 tcccagcact ttgggaggcc aagacgggca gatcacttga agtcaggagaccagcctggc 19560 caacatggca aaacctcctc tctactaaaa atacaaaaaa actagatgggtgtggtggct 19620 ggagcctgta attctagcta ctcaggaggc tgaagcagga gaatcgctgaaacccaggag 19680 gctgagattg cagtgagcca agattgcgcc actgcactgc agcctgggaaaaaaaaaaaa 19740 aattcgtagt gaaggatctg aatgacttcc agagatctgg ctctagagtatgggctcttg 19800 accgttaggt tatgctgccc cttagaaaac ttatatgctt atgttttataacaaataaat 19860 gtttagctga tttattgatt tgtttatggt ggaagtggaa gagtaagcagggcctggggc 19920 cggggctggg gccatagacc tctgcaggct ggatttttag aattcaggatccctgtcagc 19980 aaatatttaa ggccagaaga gggttgagac catctgcctt atagggttatttagcttaat 20040 ttctgtctgt atcattataa tcctaactta tgatatttta atcttctaacttcatggtct 20100 atatttcctg aaacattttt gataaattct tttgtttcta cagaaactcaaacagaagaa 20160 tcaacagctg aaacaaatta tggatcaatt acgaaatctc atctgggatataaatgccat 20220 gttggcaatg aggaactaag ctgatattta aatttcctgc tttacacatgttataccatt 20280 gttttttccc tcaagtattt tttccctgtg aagaagatta tttatctgcttttattttag 20340 tcactaaaac taaagttttt atttttacat tgtgattttt acattaaaatattaactttt 20400 ttaatgctat tttatgaaag attattgtaa taaactttga tggggtttgtattttggtta 20460 atcttcatga attgaataat tgttttttta aagcaaaata aagttttttaaataaatgtt 20520 aatatttgat taatggattt actcattcca attacccttt tagaagaaaaatatttaaat 20580 atttctgcag ataaaagt 20598

What is claimed is:
 1. An isolated polypeptide comprising at least ninecontiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4.
 2. Theisolated polypeptide of claim 1 having from 15 to 1500 amino acidresidues.
 3. The isolated polypeptide of claim 2, wherein said at leastnine contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4 areoperably linked via a peptide bond or polypeptide linker to a secondpolypeptide selected from the group consisting of maltose bindingprotein, an immunoglobulin constant region, a polyhistidine tag, and apeptide as shown in SEQ ID NO:7.
 4. The isolated polypeptide of claim 1comprising at least 30 contiguous residues of SEQ ID NO:2 or SEQ IDNO:4.
 5. The isolated polypeptide of claim 1 comprising: residues 41-55of SEQ ID NO:2 residues 56-77 of SEQ ID NO:2; residues 78-92 of SEQ IDNO:2; residues 78-92 of SEQ ID NO:4; residues 93-110 of SEQ ID NO:2;residues 111-125 of SEQ ID NO:2; residues 111-125 of SEQ ID NO:4;residues 126-148 of SEQ ID NO:2; residues 126-148 of SEQ ID NO:4; orresidues 149-163 of SEQ ID NO:2.
 6. The isolated polypeptide of claim 1comprising: residues 41-163 of SEQ ID NO:5; residues 34-163 of SEQ IDNO:5; residues 34-178 of SEQ ID NO:5; or residues 18-178 of SEQ ID NO:5.7. The isolated polypeptide of claim 1 comprising: residues 41-163 ofSEQ ID NO:2 or SEQ ID NO:4; residues 34-163 of SEQ ID NO:2 or SEQ IDNO:4; residues 34-178 of SEQ ID NO:2 or SEQ ID NO:4; or residues 18-178of SEQ ID NO:2 or SEQ ID NO:4.
 8. An expression vector comprising thefollowing operably linked elements: a transcription promoter; a DNAsegment encoding a polypeptide comprising a sequence of amino acidresidues selected from the group consisting of: residues 41-55 of SEQ IDNO:2 residues 56-77 of SEQ ID NO:2; residues 78-92 of SEQ ID NO:2;residues 78-92 of SEQ ID NO:4; residues 93-110 of SEQ ID NO:2; residues111-125 of SEQ ID NO:2; residues 111-125 of SEQ ID NO:4; residues126-148 of SEQ ID NO:2; residues 126-148 of SEQ ID NO:4; and residues149-163 of SEQ ID NO:2; and a transcription terminator.
 9. Theexpression vector of claim 8 wherein the DNA segment comprisesnucleotides 52 to 534 of SEQ ID NO:6.
 10. The expression vector of claim8 wherein the polypeptide comprises: residues 41-163 of SEQ ID NO:2 orSEQ ID NO:4; residues 34-163 of SEQ ID NO:2 or SEQ ID NO:4; residues34-178 of SEQ ID NO:2 or SEQ ID NO:4; or residues 18-178 of SEQ ID NO:2or SEQ ID NO:4.
 11. The expression vector of claim 8 further comprisinga secretory signal sequence operably linked to the DNA segment.
 12. Anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide comprisinga sequence of amino acid residues selected from the group consisting of:residues 41-163 of SEQ ID NO:5; residues 34-163 of SEQ ID NO:5; residues34-178 of SEQ ID NO:5; or residues 18-178 of SEQ ID NO:5; and atranscription terminator.
 13. The expression vector of claim 12 furthercomprising a secretory signal sequence operably linked to the DNAsegment.
 14. A cultured cell into which has been introduced theexpression vector of claim 8, wherein said cell expresses said DNAsegment.
 15. The cell of claim 14 wherein the polypeptide comprisesresidues 41-163 of SEQ ID NO:2 or residues 41-163 of SEQ ID NO:4. 16.The cell of claim 14 wherein the polypeptide comprises residues 18-178of SEQ ID NO:2 or residues 18-178 of SEQ ID NO:4.
 17. The cell of claim14 wherein the expression vector further comprises a secretory signalsequence operably linked to the DNA segment, and wherein the polypeptideis secreted by the cell.
 18. A cultured cell into which has beenintroduced the expression vector of claim 12, wherein said cellexpresses said DNA segment.
 19. The cell of claim 18 wherein theexpression vector further comprises a secretory signal sequence operablylinked to the DNA segment, and wherein the polypeptide is secreted bythe cell.
 20. A method of making a polypeptide comprising: culturing acell into which has been introduced the expression vector of claim 14under conditions whereby the DNA segment is expressed and thepolypeptide is produced; and recovering the polypeptide.
 21. The methodof claim 20 wherein the expression vector further comprises a secretorysignal sequence operably linked to the DNA segment, and wherein thepolypeptide is secreted by the cell and recovered from a medium in whichthe cell is cultured.
 22. A polypeptide produced by the method of claim20.
 23. An antibody that specifically binds to the polypeptide of claim22.
 24. A method of detecting, in a test sample, the presence of anantagonist of zalpha33 activity, comprising: culturing a cell that isresponsive to zalpha33; exposing the cell to a zalpha33 polypeptide inthe presence and absence of a test sample; comparing levels of responseto the zalpha33 polypeptide, in the presence and absence of the testsample, by a biological or biochemical assay; and determining from thecomparison the presence of an antagonist of zalpha33 activity in thetest sample.
 25. An isolated polypeptide comprising residues 18-178 ofSEQ ID NO:2.
 26. The polypeptide of claim 25, which is not more that1200 amino acid residues in length.
 27. The polypeptide of claim 26,wherein said residues 18-178 of SEQ ID NO:2 are operably linked via apeptide bond or polypeptide linker to a second polypeptide selected fromthe group consisting of maltose binding protein, an immunoglobulinconstant region, a polyhistidine tag, and a peptide as shown in SEQ IDNO:7.
 28. The polypeptide of claim 25, wherein said polypeptide consistsof residues 18-178 of SEQ ID NO:2.
 29. The polypeptide of claim 25,wherein said polypeptide comprises residues 1-178 of SEQ ID NO:2. 30.The polypeptide of claim 29, wherein said polypeptide consists ofresidues 1-178 of SEQ ID NO:2.
 31. The polypeptide of claim 25, whereinsaid polypeptide is conjugated to a detectable molecule selected fromthe group consisting of radionuclides, enzymes, substrates, cofactors,fluorescent markers, chemiluminescent markers, and magnetic particles.32. The polypeptide of claim 25, wherein said polypeptide is conjugatedto a cytotoxin.
 33. A composition comprising: a polypeptide comprisingresidues 18-178 of SEQ ID NO:2; and a pharmaceutically acceptablevehicle.